Treatment of angiogenesis disorders

ABSTRACT

This invention concerns pathological angiogenesis and cancer, related treatment methods, and related compositions. Also disclosed are related diagnosis kits and methods.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.61/441,738, filed on Feb. 11, 2011. The content of the application isincorporated herein by reference in its entirety.

FIELD OF INVENTION

This invention relates to treatments for angiogenesis disorders.

BACKGROUND OF INVENTION

Angiogenesis is a process of growth of new blood vessels and remodelingof preexisting blood vessels. It is vital for normal growth anddevelopment, as well as other physiological processes, such as woundhealing. On the other hand, angiogenesis is also important in variouspathological processes. For example, pathological angiogenesis is afundamental step in the transition of tumors from a dormant state to amalignant one, characterized by the properties of anaplasia,invasiveness, and metastasis.

Metastatic progression of cancer is a daunting clinical challenge.Technological advances have allowed for the detection and treatment ofsome early stage neoplasm, however, total death rates from epithelialmalignancies have remained essentially unchanged over the last fortyyears (seer.cancer.gov/csr/1975_(—)2007/, National Institute of Health,2007). It generally is believed that this is due to several factors,including molecular heterogeneity within cancer types, chemotherapeuticregimens of modest efficacy that were historically empirically derived,and a long-standing focus on the molecular drivers of primary tumorgrowth rather than metastatic progression.

Effective prevention or treatment of metastasis calls for understandingof molecular and cellular events, including angiogenesis, underlyingthis complex process (Talmadge, J. E. et al., Cancer Res 70 (14), 5649(2010); Sleeman, J. et al., Eur J Cancer 46 (7), 1177 (2010); and Hurst,D. R., et al., Cancer Res 69 (19), 7495 (2009)). VEGF has beendiscovered as a promoter of tumorigenesis in primary tumors (Kim, K. J.et al., Nature 362 (6423), 841 (1993)). Clinical trials have shown thatVEGF inhibition can, in combination with chemotherapy, lengthen survivalby 2-3 months in patients with stage IV colorectal or lung cancer(Hurwitz, H. et al., N Engl J Med 350 (23), 2335 (2004); Giantonio, B.J. et al., J Clin Oncol 25 (12), 1539 (2007); and Sandler, A. et al., NEngl J Med 355 (24), 2542 (2006)). However, VEGF inhibition has notproven beneficial for metastasis prevention in the adjuvant setting(Barugel, M. E., et al. Expert Rev Anticancer Ther 9 (12), 1829 (2009)and in recent pre-clinical metastasis models (Paez-Ribes, M. et al.,Cancer Cell 15 (3), 220 (2009) and Ebos, J. M. et al., Cancer Cell 15(3), 232 (2009)). While compensation by other unknown factors thatpromote metastatic angiogenesis has been proposed to underlie theseoutcomes, a number of investigators have sought to address metastasisvia pathways other than angiogenesis. For example, WO 2009082744described genes over-expressed in bone and lung metastases of breastcancer, where the genes were not related to angiogenesis. Othersendeavored to identify factors that mediate metastatic angiogenesis.Yet, the success has been limited.

Thus, there is a need for agents and methods for regulating angiogenesisand for treating disorders characterized by pathological angiogenesis,including cancer.

SUMMARY OF INVENTION

This invention is based, at least in part, on an unexpected discovery ofa new pathway that regulates endothelial recruitment and, in turnangiogenesis.

Accordingly, one aspect of this invention features a method forinhibiting endothelial recruitment, as well as angiogenesis, in asubject in need thereof. The method includes a step of administering tothe subject a first agent that inhibits expression or activity of afirst protein selected from the group consisting of IGFBP2, MERTK, andPITPNC1. In one embodiment, the subject has an angiogenesis disorder,i.e., a disorder characterized by pathological angiogenesis, such ascancer, an eye disorder, or an inflammatory disorder. Examples of thecancer include metastatic cancer. The above-mentioned method can furtherinclude a step of administering to the subject a second agent thatinhibits expression or activity of a second protein selected from thegroup consisting of IGFBP2, IGF1, IGF1R, MERTK, PITPNC1, ABCB9, PSAT1,PYGB, SHMT2, and VIPR. The aforementioned first agent or second agentcan be an antibody (or an antigen-binding portion thereof.), a nucleicacid, a polypeptide, or a small molecule compound. In one example, theabove antibody is an monoclonal that contains a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 9 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:10 shown below.

In a second aspect, this invention features a method for treatingmetastatic cancer in a subject in need thereof. The method includes astep of administering to the subject a first agent that inhibitsexpression or activity of a first protein selected from the groupconsisting of IGFBP2, MERTK, and PITPNC1, where the first agent inhibitsangiogenesis. Examples of the cancer include breast cancer. The methodcan further include a step of administering to the subject a secondagent that inhibits expression or activity of a second protein selectedfrom the group consisting of IGFBP2, IGF1, IGF1, MERTK, PITPNC1, ABCB9,PSAT1, PYGB, SHMT2, and VIPR. The first agent or second agent can be anantibody (or an antigen-binding portion thereof.), a nucleic acid, apolypeptide, or a small molecule compound. In one example, the aboveantibody is an monoclonal that contains a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 9 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 10shown below.

In a third aspect, this invention features an isolated nucleic acidhaving a sequence encoding an RNAi agent capable of inhibitingexpression of a protein selected from the group consisting of IGFBP2,MERTK, and PITPNC1. In one embodiment, the RNAi agent has adouble-stranded structure having a first strand and a second strand;each of the first and second strands is between 19 and 30 nucleotideslong; and the first strand is encoded by any one of SEQ ID NOs: 1-6 aslisted in Table 5 below.

In a fourth aspect, this invention provides a composition having anagent that inhibits expression or activity of a protein selected fromthe group consisting of IGFBP2, MERTK, and PITPNC1, where the agent canbe an antibody (or an antigen-binding portion thereof.), a nucleic acid,a polypeptide, or a small molecule compound. In one example, the agentis the above-mentioned isolated nucleic acid. In another, the agent isan antibody or an antigen-binding portion thereof.

In a fifth aspect, this invention features a method for diagnosing ametastatic potential of cancer in a subject. The method includes stepsof obtaining a first expression level for a first gene of the subjectselected from the group consisting of IGFBP2, MERTK, and PITPNC1, andcomparing the first expression level with a first predetermined levelfor the selected first gene. The subject is determined to have or beprone to develop metastatic cancer if the first expression level isgreater than the first predetermined level. The first predeterminedlevel can be obtained from a control subject that is free of cancer. Inone example, the method further includes steps of obtaining a secondexpression level for a second gene of the subject selected from thegroup consisting of IGFBP2, IGF1, IGF1R, MERTK, PITPNC1, ABCB9, PSAT1,PYGB, SHMT2, and VIPR; and comparing the second expression level with asecond predetermined level for the selected second gene. The subject isdetermined to have or be prone to develop metastatic cancer if both thefirst expression level and the second expression level are greater thanthe first predetermined level and the second predetermined level,respectively. The second predetermined level can also be obtained from acontrol subject that is free of cancer.

The invention also features a method for inhibiting endothelialrecruitment in a subject in need thereof. The method includes a step ofadministering to the subject a first agent that increases expression oractivity of GAS6 (i.e. an activating agent of GAS6). The inventionfurther features a composition having an agent that increases expressionor activity of GAS6. In one example, the aforementioned agent has GAS6activity. In another, the agent is an antibody (or an antigen-bindingportion thereof), a nucleic acid, a polypeptide, or a small moleculecompound. In one embodiment, the agent is a polypeptide having thesequence of GAS6.

In a yet another aspect, the invention features a kit for diagnosing ametastatic potential of cancer in a subject. The kit includes a firstreagent that specifically binds to a first expression product (e.g.,polypeptide or mRNA) of a first gene selected from the group consistingof IGFBP2, MERTK, and PITPNC1. The kit can further include a secondreagent that specifically binds to a second expression product of asecond gene selected from the group consisting of IGFBP2, IGF1, IGF1R,MERTK, PITPNC1, ABCB9, PSAT1, PYGB, SHMT2, and VIPR.

In a further aspect, the invention features a method for identifyinggenes and non-coding RNAs that regulate metastatic cancer colonizationof any body tissue. The method includes a first step of generating apopulation of mammalian cancer cells with increased metastatic tissuecolonization potential by performing serial rounds of a) transplantationof a population of labeled or unlabeled cancer cells into any livingtissue of the body and then b) performing isolation of said labeledcancer cells from the tissue after metastatic colonization has occurredand then c) performing repeat transplantation of isolated labeled cancercells into living tissue of the body. By performing serial rounds oftransplantation, isolation, and repeat transplantation of labeled cancercells as described above, a population of labeled or unlabeled cancercells with high metastatic tissue colonization potential is generated.The second step of the method includes transducing, transfecting, orotherwise introducing a population of one or more shRNA molecules intothe population of cancer cells with high metastatic tissue colonizationpotential to generate a population of engineered cancer cells with highmetastatic potential that express one or more shRNA molecules thatreduce expression of one or more genes or non-coding RNAs. This thepopulation of engineered cancer cells with high metastatic potentialthat express one or more shRNA molecules is then a) transplanted intoany living tissue and then b) isolated from the living tissue aftermetastatic colonization has occurred. The presence, absence, orabundance of one or more of the transfected, transduced, or otherwiseintroduced shRNAs in the population of isolated post-transplantengineered cancer cells is then assessed by either microarray analysis,DNA sequencing technology, deep sequencing technology, or cloning. Thereduction in levels of any single shRNA in the population of isolatedcells relative to its representation prior to injection indicates thatthe shRNA's target gene is required for metastatic colonization of thetissue. The increase in levels of any single shRNA in the population ofisolated cells relative to its representation prior to injectionindicates that the shRNA's target antagonizes metastatic colonization ofthe tissue. The second step of this method could also includetransducing, transfecting, or otherwise introducing a population of oneor more RNAi molecules, microRNAs, or non-coding RNAs. Additionally, thesecond step could also include transducing, transfecting, or otherwiseintroducing a population of one or more sequences encoding proteincoding genes. The population of engineered cancer cells with highmetastatic potential that express one or more protein coding genes isthen a) transplanted into any living tissue and then b) isolated fromthe living tissue after metastatic colonization has occurred. Thepresence, absence, or abundance of one or more of the transfected,transduced, or otherwise introduced coding genes in the population ofisolated post-transplant engineered cancer cells is then assessed byeither microarray analysis, DNA sequencing technology, deep sequencingtechnology, or cloning. The increase in the levels of any single gene inthe population of isolated cells relative to its representation prior toinjection indicates that the gene represents a target gene required formetastatic colonization of the tissue. The decrease in the levels of anysingle gene in the population of isolated cells relative to itsrepresentation prior to injection indicates that the gene represents atarget gene that antagonizes metastatic colonization of the tissue.

In a further aspect, the invention features a monoclonal antibody (e.g.,a humanized or human monoclonal antibody) or an antigen-binding portionthereof that neutralizes IGFBP2 function by inhibiting IGFBP2 binding toIGF1. This antibody is capable of inhibiting endothelial recruitment bycancer cells, such as metastatic breast cancer cells or inhibitingpathological angiogenesis. This antibody is also capable of inhibitingtumor progression or tumor metastasis of cancer cells, such as humanbreast cancer, in vivo. In one example, the monoclonal antibody containsa heavy chain variable region comprising the amino acid sequence of SEQID NO: 9 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 10 shown below.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1 a-f are diagrams and photographs showing that endogenous miR-126suppresses metastatic colonization. a, Bioluminescence imaging of lungmetastasis by poorly metastatic breast cancer cells upon miR-126inhibition. 4×10⁴ MDA-MB-231 cells expressing a short hairpin (miR-Zip)targeting miR-126 or the control hairpin were injected intravenouslyinto immunodeficient NOD-SCID mice. Representative mice shown correspondto the MDA-MB-231/miR-126 KD set (top) and MDA-MB-231/scrambled set(bottom) at day 49. Lung colonization was quantified throughbioluminescence imaging. n=5; error bars represent s.e.m.; p-value basedon a one-sided student's t-test at day 49. Lungs were extracted at day49 and immuno-histochemically stained for human vimentin (right). b,Bioluminescence imaging of systemic metastasis by poorly metastaticbreast cancer cells with inhibited miR-126 expression. 4×10⁴ MDA-MB-231cells expressing a short hairpin targeting miR-126 or the controlhairpin were injected via intracardiac route into athymic nude mice.Representative mice shown correspond to the MDA-MB-231/miR-126 KD set(top) and MDA-MB-231/scrambled set (bottom) at day 34. Whole bodycolonization was measured by bioluminescence and quantified. n=4; errorbars represent s.e.m.; p-value based on a one-sided student's t-test atday 34. c, The total number of metastatic foci were counted in miceinjected intracardiac with MDA-MB-231/miR-126 KD andMDA-MB-231/scrambled cells (top). Representative images of bone andbrain metastatic nodules are shown (bottom). d, 5×10⁵ MDA-MB-231 cellsexpressing a short hairpin targeting miR-126 or the control hairpin wereinjected into the mammary fat pads of immunodeficient mice. Tumorvolumes were measured over time. n=15; error bars indicate s.e.m.;p-values based on a one-sided student's t-test at day 35. e, Extractedlungs from (a) were stained for human-vimentin and the size of eachmetastatic nodule was measured through image analysis using ImageJ. f,4×10⁴ Lm2 cells expressing a doxycycline inducible pre-miR-126 cassettewere injected via the tail vein into NOD-SCID mice at day 0. At day 3,doxycycline (2 mg/ml) and sucrose (5%) were added to the drinking waterin one group of mice and only 5% sucrose in the other. At day 48, thelungs were removed and immunohistochemically stained for human vimentin(right). Total number of nodules in each lung is shown to the left.

FIGS. 2 a-2J are diagrams and photographs showing that endogenousmiR-126 non-cell autonomously suppressed metastatic angiogenesis bymetastatic breast cancer cells. a, Lung sections from FIG. 1 a werehistologically double-stained for human vimentin and MECA-32 or b, forvimentin and intravenously injected lectin. The border of each nodulewas demarcated based on vimentin staining and the lectin/MECA-32staining inside the metastatic nodule highlighted in black (lowerpanels). The area positive for lectin/MECA-32 staining within eachnodule was then determined by using ImageJ software and presented as thearea covered by lectin/MECA-32 stain per area of the given nodule (%vessel density). The distribution of % vessel density between theinjected MDA-MB-231 control and miR-126 KD cells are shown in acumulative fraction plot. n=8/group (resulting in a total 18 metastaticnodules in the control, and 68 in the miR-126 KD cells), p-value basedon the Kolmogorov-Smirnov test. c, 5×10⁴ LM2 cells expressing miR-126 ora control hairpin were seeded onto a HUVEC monolayer and adhesion wasquantified. Images of cells that had adhered to the HUVEC monolayer wereobtained and analyzed using ImageJ software. n=4; error bars represents.e.m. d, Conditioned media from 5×10⁵ LM2 cells expressing miR-126 or acontrol hairpin was obtained by incubating the cells with EGM-2 mediafor 24 h. 2.5×10⁴ HUVEC cells were seeded in triplicate, grown in theconditioned media and viable cells were counted at 5 days post seeding.n=3; error bars represent s.e.m. e, 2×10⁴ HUVEC cells were mixed with1×10⁴ LM2 cells that were transduced with miR-126 or a control hairpin,and tube formation by the HUVEC cells was assayed. Images of each wellwere obtained and the number of branch points in each image wasquantified using MetaMorph software. n=3; error bars represent s.e.m.Scale bar represents 250 μm. f, 2.5×10⁴ MDA-MB-231 cells and LM2 cellswere seeded in quadruplicate. Trans-well migration of 5×10⁴ HUVEC cellstowards the cancer cells was then assessed by counting the number ofcells that had migrated to the basal side of the trans-well inserts inimages obtained using ImageJ. n=4; error bars represent s.e.m., p-valueswere obtained using student's t-test. g, LM2 cells expressing miR-126 orthe control hairpin, as well as MDA-MB-231 cells expressing a shorthairpin targeting miR-126 or the control hairpin were subjected to theHUVEC recruitment assay. Images of the basal side of the inserts wereobtained and cells counted using ImageJ software. n=4; error barsrepresent s.e.m., p-values were obtained using student's t-test.Representative images shown correspond to the LM2/miR-126OE or controlset (top) and MDA-MB-231/miR-126 KD or control set (bottom). Scale barrepresents 100 μm. h, CN34 Parental cells and CN34 LM1a cells weresubjected to the HUVEC recruitment assay. n=4; error bars represents.e.m., p-values obtained using student's t-test. i, CN34 LM1a cellsexpressing miR-126 or the control hairpin, as well as CN34 Parentalcells expressing a short hairpin targeting miR-126 or the controlhairpin, were subjected to the HUVEC recruitment assay. n=4; error barsrepresent s.e.m., p-values obtained using student's t-test.Representative images are shown. Scale bar represents 100 μm. j, 5×10⁵Lm2 cells over-expressing miR-126 or the control hairpin, as well as5×10⁵ MDA-MB-231 cells expressing a short hairpin targeting miR-126 orthe control hairpin were mixed 1:1 with matrigel and injected into themammary fat pad. Size-matched tumors were analyzed for blood vesseldensity by immuno-histochemical staining for MECA-32. 5 individualfields were taken for each tumor and the percentage of each fieldcovered by a thresholded MECA-32 staining are given as % vessel density.Quantification is shown on top and representative images shown below.n=4; error bars represent s.e.m., p-values obtained using student'st-test.

FIGS. 3 a-3 f are diagrams and photographs showing systematicidentification of a miR-126 regulatory network that mediates metastaticendothelial recruitment. a, The miR-126 metastasis signature iscomprised of genes over-expressed in metastatic cells, down-regulated bymiR-126OE, and induced by miR-126 KD. The heatmap representsvariance-normalized expression levels based on microarray and qPCRanalyses. Colourmap corresponds to standard deviations change from themean. b-d, Kaplan-Meier curves for the (b) UCSF breast cancer cohort(117 tumors), (c) NKI cohort (295 tumors), and (d) the combinedNKI/MSK/UCSF cohort (494 tumors) depicting metastasis-free-survival ofthose patients whose primary cancers over-expressed the miR-126 eightgene signature (positive) and those that did not (negative). P-valuesbased on the Mantel-Cox log-rank test. e, Luciferase reporter assays ofmiR-126 metastasis genes in MDA-MB-231 cells expressing a short hairpintargeting miR-126 or the control KD hairpin. Reporter constructscontaining the luciferase gene upstream of the 3′UTR or coding sequences(CDS) of each miR-126 regulated gene were transfected into the variouscell lines and luciferase activity was assayed at 30 hours posttransfection. n=4; error bars represent s.e.m.; p-values were obtainedusing student's t-test. f, The miR-126 complementary regions of the3′UTR/CDS constructs were mutated and the luciferase reporter assay wasrepeated with these constructs in MDA-MB-231 cells expressing a shorthairpin targeting miR-126 or the control hairpin (right). n=4; errorbars represent s.e.m.; p-values were obtained using student's t-test.

FIGS. 4 a-4 e are a set of diagrams and photographs showing that IGFBP2,PITPNC1 and MERTK promoted metastatic colonization and angiogenesis. a,2.5×10⁴ LM2 cells expressing hairpins targeting IGFBP2, MERTK, PITPNC1,SHMT2 or the control hairpin were seeded in quadruplicate. Trans-wellmigration of 5×10⁴ HUVEC cells towards the cancer cells was thenassessed. Images of cells that migrated through the trans-well insertswere obtained and analyzed using ImageJ software. n=4; error barsrepresent s.e.m., p-values obtained using a student's t-test.Representative images are shown. Scale bar represents 100 μm. b, Therelative expression levels of IGFBP2, MERTK or PITPNC1 in human breasttumor samples from stage I/II (n=53) as compared to stage III (n=29) orstage IV (n=9) patients were quantified from the OriGene TissueScanBreast Cancer arrays using qPCR. Error bars represent s.e.m., p-valuesobtained using student's t-test. c-e, Bioluminescence imaging of lungmetastasis by lung metastatic breast cancer cells with inhibitedexpression of the various miR-126 regulated genes. 4×10⁴ LM2 cellsexpressing the control hairpin or independent short hairpins targetingIGFBP2 (c), PITPNC1 (d) and MERTK (e) were injected intravenously intoimmunodeficient NOD-SCID mice. Lung colonization was measured bybioluminescence imaging and quantified. n=5; error bars represents.e.m.; p-value based on a one-sided student's t-test.

FIGS. 5 a-5 g are diagrams and photographs showing that IGFBP2 mediatedendothelial recruitment by activating IGF1/IGF1R signaling inendothelial cells. a, IGFBP2 levels in conditioned media from HUVECrecruitment assays using MDA-MB-231 cells and LM2 cells (FIG. 2 f) werequantified by ELISA. n=4; error bars represent s.e.m., p-values obtainedusing student's t-test. b, 2.5×10⁴ MDA-MB-231 cells expressing a shorthairpin targeting miR-126 or the control hairpin, as well as LM2 cellsexpressing miR-126 or the control hairpin, were seeded in quadruplicate.Trans-well recruitment of 5×10⁴ HUVEC cells in the presence of 50 ng/mlIGFBP2 Ab or 50 ng/ml control IgG Ab towards the cancer cells was thenassessed. Images of the basal side of the trans-well inserts wereobtained and the number of cells that had migrated was quantified usingImageJ. n=4; error bars represent s.e.m., p-values obtained usingstudent's t-test. Scale bar represents 100 μm. c, 2.5×10⁴ CN34 Par cellsexpressing a short hairpin targeting miR-126 or the control hairpin, aswell as CN34 Lm1a cells expressing miR-126 or the control hairpin, wereseeded in quadruplicate. Trans-well migration of 5×10⁴ HUVEC cells inthe presence of 50 ng/ml IGFBP2 Ab or 50 ng/ml control IgG Ab towardsthe cancer cells was then assessed. n=4; error bars represent s.e.m.,p-values obtained using student's t-test. d, e, Trans-well recruitmentof HUVEC cells incubated with 20 μg/ml IGF-1 blocking Ab (d), 40 μg/mlIGF-2 blocking Ab (d), 20 μg/ml IGF1R blocking Ab (e), 5 μg/ml IGF2Rblocking Ab (e), or control IgG (d,e) towards MDA-MB-231 cellsexpressing a short hairpin targeting miR-126 or control hairpin wasassayed. n=4; error bars represent s.e.m., p-values obtained usingstudent's t-test. f, HUVEC and cancer cells were pretreated with 20μg/ml IGF1R blocking Ab or control IgG Ab for 1 hour before trans-wellrecruitment of HUVEC cells towards cancer cells were assessed. n=4;error bars represent s.e.m., p-values obtained using student's t-test g,IGFBP2 gradient was simulated by mixing the given amounts of recombinantIGFBP2 protein with matrigel (1:1) in the bottom of a well. Chemotaxisof 1.5×10⁵ HUVEC cells along the IGFBP2 gradient was then assessed bycounting the number of cells that had migrated to the basal side oftrans-well inserts after 20 h using ImageJ software. n=4; error barsrepresent s.e.m., p-values obtained using student's t-test.

FIGS. 6 a-6 e are diagrams and photographs showing that MERTK mediatedrecruitment through GAS6. a, IGFBP2 levels in conditioned media from Lm2cells expressing control hairpin or 2 independent hairpins againstPITPNC1 as determined by ELISA. b, 2.5×10⁴ MDA-MB-231 cells expressing acontrol hairpin or a hairpin targeting miR-126 were seeded inquadruplicate. Trans-well migration of 5×10⁴ HUVEC cells towards thecancer cells in the presence of 1 ng/ml GAS6 and/or 10 μg/ml MerFc wasthen assessed by counting the number of cells that had migrated to thebasal side of porous inserts in images obtained using ImageJ. n=4; errorbars represent s.e.m., p-values were obtained using student's t-test. c,IGFBP2 gradient in the presence of Gas6 and secreted MERTK was simulatedby mixing rhIGFBP2 (520 ng), Gas6 (5 ng) and MerFc (10 ug) protein withmatrigel (1:1) in the bottom of a well. Chemotaxis of 1.5×10⁵ HUVECcells along the gradient was then assessed by counting the number ofcells that had migrated to the basal side of trans-well inserts after 20h using ImageJ software. n=4; error bars represent s.e.m., p-valuesobtained using student's t-test. d, Lung sections were double stainedfor vimentin and MECA-32. The border of each nodule was drawn based onhuman-vimentin staining and MECA-32 staining inside the metastaticnodule highlighted in black (lower panels). The area positive forMECA-32 staining within each nodule was then determined by using ImageJand presented as the area covered by MECA-32 staining per area of thegiven nodule (% vessel density). The distribution of % vessel densitybetween the injected LM2 cells expressing hairpins targeting IGFBP2,PITPNC1, MERTK or a control hairpin are shown in a cumulative fractionplot. n=4, p-value based on the Kolmogorov-Smirnov Test. e, Schematic ofmiR-126 regulation of endothelial recruitment and metastaticcolonization through interaction with IGFBP2, PITPNC1 and MERTK.

FIG. 7 is a diagram showing that miR-Zip miRNA-anti-sense shRNA systemstably inhibited miR-126 expression in MDA-MB-231 cells. MDA-MB-231cells were transduced with lentivirus expressing either a miR-Zipconstruct that targets miR-126 or a scrambled version of the constructthat does not target any known microRNA (SYSTEM BIOSCIENCES, MountainView, Calif.). The expression levels of mature miR-126 were then testedusing qPCR.

FIG. 8 is a set of photographs and a diagram showing that breast cancercell-expressed miR-126 regulates perfusion in metastatic nodules. 4×10⁴MDA-MB-231 cells expressing a short hairpin targeting miR-126 or thecontrol hairpin were injected intravenously into immunodeficientNOD-SCID mice. At day 59, FITC labeled low molecular weight dextran(10.000 MW) solution was injected intravenously. The dextran moleculeswere allowed to circulate for 15 min before mice were euthanized and thelungs excised. Frozen section were prepared, and stained for humanVimentin in order to localize metastatic nodules, and the FITC signalinside the nodules was quantified with a constant threshold usingImageJ. n=5; error bars represent s.e.m., p-values obtained usingstudent's t-test.

FIGS. 9 a-9 b are a set of diagrams showing that endogenous miR-126 didnot suppress endothelial adhesion, proliferation, or tube formation. a,5×10⁴ MDA cells expressing miR-126 KD or control KD vector were seededonto a HUVEC monolayer and adhesion was assessed. Images of cells thathad adhered to the HUVEC monolayer were obtained and analyzed usingImageJ software. n=4; error bars represent s.e.m. b, Conditioned mediafrom 5×10⁵ MDA miR-126 KD or MDA control KD cells was obtained byincubating the cells with EGM-2 media for 24 h. 2.5×10⁴ HUVEC cells wereseeded in triplicate, grown in the conditioned media and viable cellswere counted at 5 days after seeding. n=3; error bars represent s.e.m.c, 2×10⁴ HUVEC cells were mixed with 1×10⁴ MDA miR-126 KD or MDA controlKD cells, and tube formation by HUVEC cells was assayed. Images of eachwell were obtained and the number of branch points in each image wasanalyzed using MetaMorph software. n=3; error bars represent s.e.m.

FIGS. 10 a-10 c are a set of diagrams and diagrams showing thatendogenous miR-126 regulated angiogenesis, but not CD45 positivelymphocyte and Mac-2 positive macrophage recruitment. a-c, 5×10⁵ MDAcells expressing control hairpin or hairpin targeting miR-126 were mixedin 1:1 ratio with Matrigel and injected in the mammary fat pad. 5 minprior to sacrifice, biotinlyated lectin was injected in the tail vein.Size matched tumors were excised and functional blood vessels weredetected by staining of the injected lectin (a), CD45⁺ lymphocytedetected by anti-CD45 (b) and Mac-2⁺ macrophages detected by anti-Mac-2(c).

FIG. 11 is a Venn diagram showing the integrative experimental path thatresulted in the identification of putative miR-126 target genes.Transcriptomic profiling of genes down-regulated by greater than 1.6fold upon miR-126 over-expression were overlapped with genesup-regulated by more than 1.4 fold in metastatic LM2 cells as comparedto the parental MDA cells. This led to the identification of 23potential miR-126 target genes. By qPCR, 8 of these 23 genes weremodulated by miR-126 in both the MDA-MB-231 breast cancer cell line andthe primary CN34 cell line. These 8 genes were functionally tested fordirect regulation by miR-126 through luciferase reporter assays.

FIGS. 12 a-12 b are diagrams showing that miR-126 regulated IGFBP2 andMERTK through 3′UTR interactions and PITPNC1 and SHMT2 through CDSinteractions. a, b, Luciferase reporter assays of the miR-126 metastasisgene set in MDA-MB-231 cells expressing a short hairpin targetingmiR-126 as well as the control KD hairpin. Reporter constructscontaining the luciferase gene upstream of the 3′UTR (a) or CDS (b) ofABCB9, IGFBP2, MERTK, PITPNC1, PSAT1, PYBG, SHMT2 and VIPR1 weretransfected into the various cell lines and luciferase activity wasassayed at 30 hours post transfection. n=4; error bars represent s.e.m.;p-values were obtained using student's t-test.

FIGS. 13 a-13 d are a set of diagrams showing that independent hairpinsdown-regulated the expression levels of IGFBP2, PITPNC1 and MERTK in LM2cells. LM2 cells were transduced with lentivirus expressing a controlhairpin or a short hairpin construct targeting IGFBP2, PITPNC1 or MERTK.The expression levels of the target genes were analyzed through qPCR.

FIG. 14 is a diagram showing proliferation analysis of miR-126 targetgenes. 2.5×10⁴ LM2 cells expressing a control hairpin or short hairpinstargeting IGFBP2, PITPNC1 or MERTK were seeded in triplicate and viablecells were counted at 5 days after seeding. n=3; error bars represents.e.m.

FIG. 15 is a diagram showing that IGFBP2 promoted HUVEC migration. HUVECcells were stimulated with the given amounts of recombinant human IGFBP2protein and anti-IGF1R Ab (10 μg/ml) for 40 min, trypsinized and 5×10⁴cells were seeded into a porous transwell insert. The cells were allowedto migrate for 24 hours before the number of cells that migrated acrossthe membrane was quantified. n=6; error bars represent s.e.m.; p-valueswere obtained using student's t-test.

FIG. 16 is a photograph of Western blot analysis of MERTK in MDA-MB-231cellular lysate and conditioned media, showing that ectodomain of MERTKwas cleaved and secreted by MDA-MB-231 cells.

FIG. 17 is a set of photographs and diagrams showing that GFBP2, PITPNC1and MERTK promoted metastatic angiogenesis. Lung sections werehistologically double-stained for human vimentin and intravenouslyinjected lectin. Nodule borders were demarcated based on vimentinstaining and lectin staining inside metastatic nodules. ImageJ was usedto determine the area positive for lectin staining within each nodule. %vessel density represents the area covered by lectin staining per areaof a given nodule. The distribution of % vessel density between theinjected LM2 cells expressing the control hairpin or short hairpinstargeting IGFBP2, PITPNC1 or MERTK are shown in a cumulative fractionplot. n=5. P-value based on the Kolmogorov-Smirnov test.

FIG. 18 is a diagram showing that endogenous miR-126 expression in HUVECcells did not suppress recruitment of other HUVEC cells. An antagomiRtargeting miR-126 or a control antagomiR were transfected into HUVECcells before being subjected to the HUVEC recruitment assay. Images ofthe basal side of the inserts were obtained and cells counted usingImageJ software. n=4; error bars represent s.e.m.

FIG. 19 is a diagram showing expression of potential miR-126 targets inHUVEC cells with suppressed miR-126 levels. An antagomiR targetingmiR-126 or a control antagomiR were transfected into HUVEC cells and therelative expression of potential targets in transfected cells werequantified using qPCR. Error bars represent s.e.m., p-values obtainedusing student's t-test.

FIG. 20 is a set of diagrams showing data from antibody-capture ELISAassays used to characterize binding properties of IGFBP2 neutralizingantibodies. The figure shows that supernatant from one of the hybridomalibraries (wo6663-1) generated from animals inoculated with recombinantIGFBP2 total peptide, contains antibodies that bind to IGFBP2 with highaffinity.

FIG. 21 is a set of diagrams showing data from antibody capture ELISAassays used to characterize binding properties of IGFBP2 neutralizingantibodies. The figure shows that supernatant from hybridoma wo6663-1contains antibodies that bind to IGFBP2 to neutralize IGF1 binding toIGFBP2. Also note that the antibodies from the hybridoma wo6663-1 bindspecifically to IGFBP2, and do not bind other IGFBP family members(IGFBP3, IGFBP4).

FIG. 22 is a set of diagrams showing data from antibody capture ELISAassays used to characterize binding properties of IGFBP2 neutralizingmonoclonal antibodies recovered from single hybridoma clones isolatedfrom hybridoma library wo6663-1. Several of the IGFBP2 neutralizingmonoclonal antibodies, including M1, M4, M6, M9, M13, M14, M15, and M16,were able to bind IGFBP2 with high affinity and neutralize its bindingto IGF1.

FIG. 23 is a diagram showing that a composition containing physiologicalconcentrations of the monoclonal antibody M14 is capable of inhibitingendothelial recruitment by metastatic breast cancer cells. As inexperiments described above, a trans-well migration assay was used toquantify endothelial recruitment by metastatic cells. Highly metastaticLM2 human breast cancer cells were placed in the bottom of a Boydenchamber, where their ability to recruit HUVECS through a poroustrans-well insert could be assayed. The addition of a small physiologicconcentration of M14 to the transwell was able to significantly inhibitthe recruitment and migration (migrated cells per field) of HUVEC cells(50% reduction in migrated cells) versus the negative controls (IgG andM5 antibodies). Error bars represent s.e.m.

FIG. 24 is a diagram showing that a composition containing physiologicalconcentrations of the monoclonal antibody M14 is capable of inhibitingbreast cancer tumor progression in vivo in a mouse model.Bioluminescence imaging of mammary tumor growth by 2000 MDA-MB-231 humanbreast cancer cells in animals treated with either PBS or monoclonalantibody M14. At day 14, tumor progression was significantly inhibitedby treatment with M14 (7 to 11 fold reduction in tumor progression)compared with the PBS treated mice. The signal is normalized to signalfrom Day 0. Significance is based on a two sided student T-test.

DETAILED DESCRIPTION OF THE INVENTION

The described invention provides reagents and methods for treatingdisorders characterized by pathological angiogenesis, such asmetastasis.

As disclosed herein, a systematic analysis and focus on metastasis andmetastatic angiogenesis led to the identification of a number ofmolecules, including secreted IGFBP2, the transferase PITPNC1, thekinase MERTK, and miR-126, as targets for therapeutic inhibition withthe potential for treating metastatic cancer. A newly discovered pathwaycoordinates IGFBP2, PITPNC1, and MERTK-pro-angiogenic genes thatcorrelate in expression with human metastasis. These genes representregulators of metastatic endothelial recruitment and angiogenesis. Forexample, IGFBP2, a protein secreted by metastatic cells, recruitsendothelia by modulating IGF1-mediated activation of the IGF type-IReceptor on endothelial cells.

Endothelial recruitment is a process where endothelial cells or theirprogenitors are mobilized and homing to a site or region in a subjectfor generating new blood vessels or remodeling of preexisting bloodvessels, i.e., angiogenesis. Inhibiting this process via theabove-mentioned new pathway can be used to inhibit pathologicalangiogenesis, and thereby to treat disorders characterized bypathological angiogenesis, such as metastasis.

To inhibit endothelial recruitment and resulting angiogenesis in asubject in need thereof, one can administer to the subject an agent thatinhibits expression or activity of a protein selected from IGFBP2, IGF1,IGF1R, MERTK, PITPNC1, ABCB9, PSAT1, PYGB, SHMT2, and VIPR. Listed beloware the amino acid sequences of these proteins. The agent can be anucleic acid, a polypeptide, an antibody, or a small molecule compound.

IGFBP2 MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ IGF1 (isoform 1)MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGEYENKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGSTFEERK IGF1 (isoform 2)MITPTVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGEYENKPTGYGSSSRRAPQTGIVDECCERSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM IGF1 (isoform 3)MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGEYENKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPGGEQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK IGF1 (isoform 4)MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGEYENKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM IGF1RMKSGSGGGSPTSLWGLLFLSAALSLWPTSGEICGPGIDIRNDYQQLKRLENCTVIEGYLHILLISKAEDYRSYRFPKLTVITEYLLLFRVAGLESLGDLFPNLTVIRGWKLFYNYALVIFEMTNLKDIGLYNLRNITRGAIRIEKNADLCYLSTVDWSLILDAVSNNYIVGNKPPKECGDLCPGTMEEKPMCEKTTINNEYNYRCWTTNRCQKMCPSTCGKRACTENNECCHPECLGSCSAPDNDTACVACRHYYYAGVCVPACPPNTYRFEGWRCVDRDFCANILSAESSDSEGFVIHDGECMQECPSGFIRNGSQSMYCIPCEGPCPKVCEEEKKTKTIDSVTSAQMLQGCTIFKGNLLINIRRGNNIASELENFMGLIEVVTGYVKIRHSHALVSLSFLKNLRLILGEEQLEGNYSFYVLDNQNLQQLWDWDHRNLTIKAGKMYFAFNPKLCVSEIYRMEEVTGTKGRQSKGDINTRNNGERASCESDVLHFTSTTTSKNRIIITWHRYRPPDYRDLISFTVYYKEAPFKNVTEYDGQDACGSNSWNMVDVDLPPNKDVEPGILLHGLKPWTQYAVYVKAVTLTMVENDHIRGAKSEILYIRTNASVPSIPLDVLSASNSSSQLIVKWNPPSLPNGNLSYYIVRWQRQPQDGYLYRHNYCSKDKIPIRKYADGTIDIEEVTENPKTEVCGGEKGPCCACPKTEAEKQAEKEEAEYRKVFENFLHNSIFVPRPERKRRDVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPFTLYRIDIHSCNHEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDPVFFYVQAKTGYENFIHLIIALPVAVLLIVGGLVIMLYVFHRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLREVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPLPQSSTC MERTKMGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRKSTAHSILISWVPGEDGYSPERNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDELLFADDSSEGSEVLM PITPNC1 (isoform a)MLLKEYRICMPLTVDEYKIGQLYMISKHSHEQSDRGEGVEVVQNEPFEDPHHGNGQFTEKRVYLNSKLPSWARAVVPKIFYVTEKAWNYYPYTITEYTCSFLPKFSIHIETKYEDNKGSNDTIFDNEAKDVEREVCFIDIACDEIPERYYKESEDPKHFKSEKTGRGQLREGWRDSHQPIMCSYKLVTVKFEVWGLQTRVEQFVHKVVRDILLIGHRQAFAWVDEWYDMTMDEVREFERATQEATNKKIGIFPPAISISSIPLLPSSVRSAPSSAPSTPLSTDAPEFLSVPKDRPRKKSAPETLTLPDPEKKATLNLPGMHSSDKPCRPKSE PITPNC1 (isoform b)MLLKEYRICMPLTVDEYKIGQLYMISKHSHEQSDRGEGVEVVQNEPFEDPHHGNGQFTEKRVYLNSKLPSWARAVVPKIFYVTEKAWNYYPYTITEYTCSFLPKFSIHIETKYEDNKGSNDTIFDNEAKDVEREVCFIDIACDEIPERYYKESEDPKHFKSEKTGRGQLREGWRDSHQPIMCSYKLVTVKFEVWGLQTRVEQFVHKVVRDILLIGHRQAFAWVDEWYDMTMDDVREYEKNMHEQTNIKVCNQHSSPVDDIESHAQTST ABCB9MRLWKAVVVTLAFMSVDICVTTAIYVFSHLDRSLLEDIRHFNIFDSVLDLWAACLYRSCLLLGATIGVAKNSALGPRRLRASWLVITLVCLFVGIYAMVKLLLFSEVRRPIRDPWFWALFVWTYISLGASFLLWWLLSTVRPGTQALEPGAATEAEGFPGSGRPPPEQASGATLQKLLSYTKPDVAELVAASFELIVAALGETFLPYYTGRAIDGIVIQKSMDQFSTAVVIVCLLAIGSSFAAGIRGGIFTLIFARLNIRLRNCLERSLVSQETSFEDENRTGDLISRLTSDTTMVSDLVSQNINVFLRNTVKVTGVVVFMFSLSWQLSLVTFMGFPIIMMVSNIYGKYYKRLSKEVQNALARASNTAEETISAMKTVRSFANEEEEAEVYLRKLQQVYKLNRKEAAAYMYYVWGSGLTLLVVQVSILYYGGHLVISGQMTSGNLIAFIIYEFVLGDCMESVGSVYSGLMQGVGAAEKVFEFIDRQPTMVHDGSLAPDHLEGRVDFENVTFTYRTRPHTQVLQNVSFSLSPGKVTALVGPSGSGKSSCVNILENFYPLEGGRVLLDGKPISAYDHKYLHRVISLVSQEPVLFARSITDNISYGLPTVPFEMVVEAAQKANAHGFIMELQDGYSTETGEKGAQLSGGQKQRVAMARALVRNPPVLILDEATSALDAESEYLIQQAIHGNLQKHTVLIIAHRLSTVEHAHLIVVLDKGRVVQQGTHQQLLAQGGLYAKLVQRQMLGLQPAADFTAGHNEPVANGSHKA PSAT1 (isoform 1)MDAPRQVVNFGPGPAKLPHSVLLEIQKELLDYKGVGISVLEMSHRSSDFAKIINNTENLVRELLAVPDNYKVIFLQGGGCGQFSAVPLNLIGLKAGRCADYVVTGAWSAKAAEEAKKEGTINIVHPKLGSYTKIPDPSTWNLNPDASYVYYCANETVHGVEFDFIPDVKGAVLVCDMSSNFLSKPVDVSKFGVIFAGAQKNVGSAGVTVVIVRDDLLGFALRECPSVLEYKVQAGNSSLYNTPPCFSIYVMGLVLEWIKNNGGAAAMEKLSSIKSQTIYEIIDNSQGFYVCPVEPQNRSKMNIPERIGNAKGDDALEKRFLDKALELNMLSLKGHRSVGGIRASLYNAVTIEDVQKLAAF MKKFLEMHQLPSAT1 (isoform 2)MDAPRQVVNFGPGPAKLPHSVLLEIQKELLDYKGVGISVLEMSHRSSDFAKIINNTENLVRELLAVPDNYKVIFLQGGGCGQFSAVPLNLIGLKAGRCADYVVTGAWSAKAAEEAKKFGTINIVHPKLGSYTKIPDPSTWNLNPDASYVYYCANETVHGVEFDFIPDVKGAVLVCDMSSNFLSKPVDVSKFGVIFAGAQKNVGSAGVTVVIVRDDLLGFALRECPSVLEYKVQAGNSSLYNTPPCFSIYVMGLVLEWIKNNGGAAAMEKLSSIKSQTIYEIIDNSQGFYVSVGGIRASLYNAVTIEDVQKLAAFMKKFLEMHQL PYGBMAKPLTDSEKRKQISVRGLAGLGDVAEVRKSFNRHLHFTLVKDRNVATPRDYFFALAHTVRDHLVGRWIRTQQHYYERDPKRIYYLSLEFYMGRTLQNTMVNLGLQNACDEAIYQLGLDLEELEEIEEDAGLGNGGLGRLAACFLDSMATLGLAAYGYGIRYEFGIFNQKIVNGWQVEEADDWLRYGNPWEKARPEYMLPVHFYGRVEHTPDGVKWLDTQVVLAMPYDTPVPGYKNNTVNTMRLWSAKAPNDFKLQDFNVGDYIEAVLDRNLAENISRVLYPNDNFFEGKELRLKQEYFVVAATLQDIIRRFKSSKFGCRDPVRTCFETFPDKVAIQLNDTHPALSIPELMRILVDVEKVDWDKAWEITKKTCAYTNHTVLPEALERWPVSMFEKLLPRHLEIIYAINQRHLDHVAALFPGDVDRLRRMSVIEEGDCKRINMAHLCVIGSHAVNGVARIHSEIVKQSVFKDFYELEPEKFQNKTNGITPRRWLLLCNPGLADTIVEKIGEEFLTDLSQLKKLLPLVSDEVFIRDVAKVKQENKLKFSAFLEKEYKVKINPSSMFDVHVKRIHEYKRQLLNCLHVVTLYNRIKRDPAKAFVPRTVMIGGKAAPGYHMAKLIIKLVTSIGDVVNHDPVVGDRLKVIFLENYRVSLAEKVIPAADLSQQISTAGTEASGTGNMKFMLNGALTIGTMDGANVEMAEEAGAENLFIFGLRVEDVEALDRKGYNAREYYDHLPELKQAVDQISSGFFSPKEPDCFKDIVNMLMHHDRFKVFADYEAYMQCQAQVDQLYRNPKENTKKVIRNIACSGKFSSDRTITEYAREIWGVEPSDLQIPPPNI PRDSHMT2 (isoform 1)MLYFSLFWAARPLQRCGQLVRMAIRAQHSNAAQTQTGEANRGWTGQESLSDSDPEMWELLQREKDRQCRGLELIASENFCSRAALEALGSCLNNKYSEGYPGKRYYGGAEVVDEIELLCQRRALEAFDLDPAQWGVNVQPYSGSPANLAVYTALLQPHDRIMGLDLPDGGHLTHGYMSDVKRISATSIFFESMPYKLNPKTGLIDYNQLALTARLFRPRLIIAGTSAYARLIDYARMREVCDEVKAHLLADMAHISGLVAAKVIPSPFKHADIVTTTTHKTLRGARSGLIFYRKGVKAVDPKTGREIPYTFEDRINFAVFPSLQGGPHNHAIAAVAVALKQACTPMFREYSLQVLKNARAMADALLERGYSLVSGGTDNHLVLVDLRPKGLDGARAERVLELVSITANKNTCPGDRSAITPGGLRLGAPALTSRQFREDDERRVVDFIDEGVNIGLEVKSKTAKLQDFKSFLLKDSETSQRLANLRQRVEQFARAFPMPGFDEH SHMT2 (isoform 2)MLYFSLFWAARPLQRCGQLVRMAIRAQHSNAAQTQTGEANRGWTGQESLSDSDPEMWELLQREKDRQCRGLELIASENFCSRAALEALGSCLNNKYSEGYPGKRYYGGAEVVDEIELLCQRRALEAFDLDPAQWGVNVQPYSGSPANLAVYTALLQPHDRIMGLDLPDGGHLTHGYMSDVKRISATSIFFESMPYKLNLALTARLFRPRLIIAGTSAYARLIDYARMREVCDEVKAHLLADMAHISGLVAAKVIPSPFKHADIVTTTTHKTLRGARSGLIFYRKGVKAVDPKTGREIPYTFEDRINFAVFPSLQGGPHNHAIAAVAVALKQACTPMFREYSLQVLKNARAMADALLERGYSLVSGGTDNHLVLVDLRPKGLDGARAERVLELVSITANKNTCPGDRSAITPGGLRLGAPALTSRQFREDDFRRVVDFIDEGVNIGLEVKSKTAKLQDFKSFLLKDSETSQRLANLRQRVEQFARAFPMPGFDEH SHMT2 (isoform 3)MAIRAQHSNAAQTQTGEANRGWTGQESLSDSDPEMWELLQREKDRQCRGLELIASENFCSRAALEALGSCLNNKYSEGYPGKRYYGGAEVVDEIELLCQRRALEAFDLDPAQWGVNVQPYSGSPANLAVYTALLQPHDRIMGLDLPDGGHLTHGYMSDVKRISATSIFFESMPYKLNPKTGLIDYNQLALTARLFRPRLIIAGTSAYARLIDYARMREVCDEVKAHLLADMAHISGLVAAKVIPSPFKHADIVTTTTHKTLRGARSGLIFYRKGVKAVDPKTGREIPYTFEDRINFAVFPSLQGGPHNHAIAAVAVALKQACTPMFREYSLQVLKNARAMADALLERGYSLVSGGTDNHLVLVDLRPKGLDGARAERVLELVSITANKNTCPGDRSAITPGGLRLGAPALTSRQFREDDFRRVVDFIDEGVNIGLEVKSKTAKLQDFKSFLLKDSETSQRLANLRQRVEQFARAFPMPGF DEH VIPR 1MRPPSPLPARWLCVLAGALAWALGPAGGQAARLQEECDYVQMIEVQHKQCLEEAQLENETIGCSKMWDNLTCWPATPRGQVVVLACPLIFKLFSSIQGRNVSRSCTDEGWTHLEPGPYPIACGLDDKAASLDEQQTMFYGSVKTGYTIGYGLSLATLLVATAILSLFRKLHCTRNYIHMHLFISFILRAAAVFIKDLALFDSGESDQCSEGSVGCKAAMVFFQYCVMANFFWLLVEGLYLYTLLAVSFFSERKYFWGYILIGWGVPSTFTMVWTIARIHFEDYGCWDTINSSLWWIIKGPILTSILVNFILFICIIRILLQKLRPPDIRKSDSSPYSRLARSTLLLIPLFGVHYIMFAFFPDNFKPEVKMVFELVVGSFQGFVVAILYCFLNGEVQAELRRKWRRWHLQGVLGWNPKYRHPSGGSNGATCSTQVSMLTRVSPGARRSSSFQAEVSLV

An inhibitory agent (i.e., inhibitor) or an activating agent (i.e.,activator) can be a nucleic acid, a polypeptide, an antibody, or a smallmolecule compound. Preferably, it is an isolated agent, but not anendogenous molecule (a micro RNA) in a cell of the subject. In oneexample, it excludes a micro RNA that is endogenous in human cells,e.g., miR-126, miR206, or/and miR-335. In another example, theinhibitory or activating agent functions at a level of transcription,mRNA stability, translation, protein stability/degradation, proteinmodification, and protein binding.

A nucleic acid refers to a DNA molecule (for example, but not limitedto, a cDNA or genomic DNA), an RNA molecule (for example, but notlimited to, an mRNA), or a DNA or RNA analog. A DNA or RNA analog can besynthesized from nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded. An “isolated nucleic acid” is anucleic acid the structure of which is not identical to that of anynaturally occurring nucleic acid or to that of any fragment of anaturally occurring genomic nucleic acid. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein.

The terms “RNA,” “RNA molecule,” and “ribonucleic acid molecule” areused interchangeably herein, and refer to a polymer of ribonucleotides.The term “DNA” or “DNA molecule” or deoxyribonucleic acid molecule”refers to a polymer of deoxyribonucleotides. DNA and RNA can besynthesized naturally (e.g., by DNA replication or transcription of DNA,respectively). RNA can be post-transcriptionally modified. DNA and RNAalso can be chemically synthesized. DNA and RNA can be single-stranded(i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g.,double-stranded, i.e., dsRNA and dsDNA, respectively).

The nucleic acid sequence can encode a small interference RNA (e.g., anRNAi agent) that targets one or more of the above-mentioned genes andinhibits its expression or activity. The term “RNAi agent” refers to anRNA, or analog thereof, having sufficient sequence complementarity to atarget RNA to direct RNA interference. Examples also include a DNA thatcan be used to make the RNA. RNA interference (RNAi) refers to asequence-specific or selective process by which a target molecule (e.g.,a target gene, protein or RNA) is down-regulated. Generally, aninterfering RNA (“iRNA”) is a double stranded short-interfering RNA(siRNA), short hairpin RNA (shRNA), or single-stranded micro-RNA (miRNA)that results in catalytic degradation of specific mRNAs, and also can beused to lower or inhibit gene expression.

The term “short interfering RNA” or “siRNA” (also known as “smallinterfering RNAs”) refers to an RNA agent, preferably a double-strandedagent, of about 10-50 nucleotides in length, preferably between about15-25 nucleotides in length, more preferably about 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length, the strands optionally havingoverhanging ends comprising, for example 1, 2 or 3 overhangingnucleotides (or nucleotide analogs), which is capable of directing ormediating RNA interference. Naturally-occurring siRNAs are generatedfrom longer dsRNA molecules (e.g., >25 nucleotides in length) by acell's RNAi machinery (e.g., Dicer or a homolog thereof).

The term “miRNA” or “microRNA” refers to an RNA agent, preferably asingle-stranded agent, of about 10-50 nucleotides in length, preferablybetween about 15-25 nucleotides in length, more preferably about 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length, which is capable ofdirecting or mediating RNA interference. Naturally-occurring miRNAs aregenerated from stem-loop precursor RNAs (i.e., pre-miRNAs) by Dicer. Theterm “Dicer” as used herein, includes Dicer as well as any Dicerorthologue or homologue capable of processing dsRNA structures intosiRNAs, miRNAs, siRNA-like or miRNA-like molecules. The term microRNA(or “miRNA”) is used interchangeably with the term “small temporal RNA”(or “stRNA”) based on the fact that naturally-occurring microRNAs (or“miRNAs”) have been found to be expressed in a temporal fashion (e.g.,during development).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region.

Thus, also within the scope of this invention is utilization of RNAifeaturing degradation of RNA molecules (e.g., within a cell).Degradation is catalyzed by an enzymatic, RNA-induced silencing complex(RISC). A RNA agent having a sequence sufficiently complementary to atarget RNA sequence (e.g., one or more of the above-mentioned genes) todirect RNAi means that the RNA agent has a homology of at least 50%,(e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% homology) to thetarget RNA sequence so that the two are sufficiently complementary toeach other to hybridize and trigger the destruction of the target RNA bythe RNAi machinery (e.g., the RISC complex) or process. A RNA agenthaving a “sequence sufficiently complementary to a target RNA sequenceto direct RNAi” also means that the RNA agent has a sequence sufficientto trigger the translational inhibition of the target RNA by the RNAimachinery or process. A RNA agent also can have a sequence sufficientlycomplementary to a target RNA encoded by the target DNA sequence suchthat the target DNA sequence is chromatically silenced. In other words,the RNA agent has a sequence sufficient to induce transcriptional genesilencing, e.g., to down-modulate gene expression at or near the targetDNA sequence, e.g., by inducing chromatin structural changes at or nearthe target DNA sequence.

The above-mentioned polynucleotides can be delivered using polymeric,biodegradable microparticle or microcapsule delivery devices known inthe art. Another way to achieve uptake of the polynucleotides is usingliposomes, prepared by standard methods. The polynucleotide can beincorporated alone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano, etal., 1995, J. Mol. Med. 73:479). Alternatively, tissue specifictargeting can be achieved by the use of tissue-specific transcriptionalregulatory elements that are known in the art. Delivery of naked DNA(i.e., without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site is another means to achieve in vivo expression.

siRNA, miRNA, and asRNA (antisense RNA) molecules can be designed bymethods well known in the art. siRNA, miRNA, and asRNA molecules withhomology sufficient to provide sequence specificity required to uniquelydegrade any RNA can be designed using programs known in the art,including, but not limited to, those maintained on websites for AMBION,Inc. and DHARMACON, Inc. Systematic testing of several designed speciesfor optimization of the siRNA, miRNA, and asRNA sequence can beroutinely performed by those skilled in the art. Considerations whendesigning short interfering nucleic acid molecules include, but are notlimited to, biophysical, thermodynamic, and structural considerations,base preferences at specific positions in the sense strand, andhomology. These considerations are well known in the art and provideguidelines for designing the above-mentioned RNA molecules.

In one example, the polypeptide is an antibody. The term “antibody”refers to an immunoglobulin molecule or immunologically active portionthereof, i.e., an antigen-binding portion. Examples include, but are notlimited to, a protein having at least one or two, heavy (H) chainvariable regions (V_(H)), and at least one or two light (L) chainvariable regions (V_(L)). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). As used herein, the term“immunoglobulin” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized human immunoglobulin genes include the kappa, lambda, alpha(IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon andmu constant region genes, as well as the myriad immunoglobulin variableregion genes.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., IGFBP2, IGF1, IGF1R,MERTK, PITPNC1, ABCB9, PSAT1, PYGB, SHMT2, or VIPR). It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi)an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

Antibodies that specifically bind to one of the above-mentioned targetprotein can be made using methods known in the art. This antibody can bea polyclonal or a monoclonal antibody. Examples of such antibodiesinclude those described in the working examples below. In oneembodiment, the antibody can be recombinantly produced, e.g., producedby phage display or by combinatorial methods. In another embodiment, theantibody is a fully human antibody (e.g., an antibody made in a mousewhich has been genetically engineered to produce an antibody from ahuman immunoglobulin sequence), a humanized antibody, or a non-humanantibody, for example, but not limited to, a rodent (mouse or rat),goat, primate (for example, but not limited to, monkey), rabbit, orcamel antibody. Examples of methods to generate humanized version ofantibodies include, but are not limited to, CDR grafting (Queen et al.,U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)), chainshuffling (U.S. Pat. No. 5,565,332); and veneering or resurfacing (EP592,106; EP 519,596); Padlan, Molecular Immunology 28(415):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)). Examples of methods togenerate fully human antibodies include, but are not limited to,generation of antibodies from mice that can express human immunoglobulingenes and use of phage-display technology to generate and screen humanimmunoglobulin gene libraries.

An “isolated antibody” is intended to refer to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically bindsIGFBP2, IGF1, IGF1R, MERTK, PITPNC1, ABCB9, PSAT1, PYGB, SHMT2, or VIPRis substantially free of antibodies that specifically bind antigensother than such an antigen). An isolated antibody that specificallybinds the antigen may, however, have cross-reactivity to other antigens,such as IGFBP2, IGF1, IGF1R, MERTK, PITPNC1, ABCB9, PSAT1, PYGB, SHMT2,or VIPR molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less,more preferably 10⁻⁹ M or less and even more preferably 10⁻¹⁰ M or lessfor a target antigen. However, “high affinity” binding can vary forother antibody isotypes. For example, “high affinity” binding for an IgMisotype refers to an antibody having a K_(D) of 10⁻⁷ M or less, morepreferably 10⁻⁸ M or less.

In one example, a composition comprising a monoclonal antibody thatneutralizes IGFBP2 function by inhibiting IGFBP2 binding to IGF1 isdescribed. In one embodiment, this antibody can be a fully humanantibody, a humanized antibody, or a non-human antibody, for example,but not limited to, a rodent (mouse or rat), goat, primate (for example,but not limited to, monkey), rabbit, or camel antibody. In oneembodiment, one or more amino-acids of this monoclonal monoclonalantibody may be substituted in order to alter its physical properties.These properties include, but are not limited to, binding specificity,binding affinity, immunogenicity, and antibody isotype. Pharmaceuticalcompositions containing fully human or humanized versions of the abovedescribed antibodies can be used to treat disorders of pathologicalangiogenesis.

In one example, a composition comprising an IGFBP2 neutralizing antibodythat inhibits IGF1 from binding to IGFBP2 inhibits breast cancer tumorprogression and tumor burden in vivo. In this example, administration ofthe above described antibody reduced tumor burden of human breast cancerin vivo in a mouse model of human cancer.

Pharmaceutical compositions containing fully human or humanized versionsof the above described antibodies can be used to inhibit breast cancermetastasis in human patients by inhibiting endothelial recruitment bymetastatic cells. In another embodiment, pharmaceutical compositionscontaining fully human or humanized versions of these antibodies can beused to treat other types of vascular tumors. Typical vascularizedtumors that can be treated with this composition include solid tumors,particularly carcinomas, which require a vascular component for theprovision of oxygen and nutrients. Exemplary solid tumors include, butare not limited to, carcinomas of the lung, breast, bone, ovary,stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliarytract, colon, rectum, cervix, uterus, endometrium, kidney, bladder,prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cellcarcinomas, melanomas, gliomas, glioblastomas, neuroblastomas, Kaposi'ssarcoma, and sarcomas.

In another embodiment, the polypeptide is a mutant form of theabove-mentioned protein, which interferes with the above-mentionedpathway and therefore inhibits endothelial recruitment and angiogenesis.The term “mutant” encompasses naturally occurring mutants and mutantscreated chemically and/or using recombinant DNA techniques. A mutant ofone of the above-mentioned wild type polypeptide can be due toalteration, e.g., truncation, elongation, substitution, deletion, orinsertion, of one or more amino acids. The alteration also can have amodified amino acid, such as one comprising a post-translationalmodification. The pro-angiogenic activity of a mutant, if any, issubstantially lower than the activity of the wild type polypeptide by atleast about 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) asmeasured using an assay described herein or known in the art. Oneexample is a polypeptide having the extracellular domain of IGF1-R, butlacking the intra-cellular domain. By competing for IGF-1, this mutantcan inhibit the above-mentioned pathway and pro-angiogenic activity in adominant-negative manner.

The amino acid compositions of the above-mentioned antibodies orpolypeptides may vary with or without disrupting the ability (e.g.,affinity) to bind to the respective antigens or targets, and trigger orinhibit the respective cellular response. For example, they can containone or more conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in, e.g., SEQ ID NO: 9 or 10 can be replaced withanother amino acid residue from the same side chain family.Alternatively, mutations can be introduced randomly along all or part ofthe sequences, such as by saturation mutagenesis, and the resultantmutants can be screened for the ability to bind to the respectiveantigen and trigger the respective cellular response to identify mutantsthat retain the activity.

Compositions

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the agents described above. Thecomposition can be a pharmaceutical composition that contains apharmaceutically acceptable carrier, a dietary composition that containsa dietarily acceptable suitable carrier, or a cosmetic composition thatcontains a cosmetically acceptable carrier.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. A “pharmaceutically acceptable carrier,” after administered to orupon a subject, does not cause undesirable physiological effects. Thecarrier in the pharmaceutical composition must be “acceptable” also inthe sense that it is compatible with the active ingredient and can becapable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active compound.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. Examples of othercarriers include colloidal silicon oxide, magnesium stearate, cellulose,sodium lauryl sulfate, and D&C Yellow #10.

The above-described composition, in any of the forms described above,can be used for treating disorders characterized by pathologicalangiogenesis. An effective amount refers to the amount of an activecompound/agent that is required to confer a therapeutic effect on atreated subject. Effective doses will vary, as recognized by thoseskilled in the art, depending on the types of diseases treated, route ofadministration, excipient usage, and the possibility of co-usage withother therapeutic treatment.

A pharmaceutical composition of this invention can be administeredparenterally, orally, nasally, rectally, topically, or buccally. Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intrmuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent. Such solutionsinclude, but are not limited to, 1,3-butanediol, mannitol, water,Ringer's solution, and isotonic sodium chloride solution. In addition,fixed oils are conventionally employed as a solvent or suspending medium(e.g., synthetic mono- or diglycerides). Fatty acid, such as, but notlimited to, oleic acid and its glyceride derivatives, are useful in thepreparation of injectables, as are natural pharmaceutically acceptableoils, such as, but not limited to, olive oil or castor oil,polyoxyethylated versions thereof. These oil solutions or suspensionsalso can contain a long chain alcohol diluent or dispersant such as, butnot limited to, carboxymethyl cellulose, or similar dispersing agents.Other commonly used surfactants, such as, but not limited to, Tweens orSpans or other similar emulsifying agents or bioavailability enhancers,which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms also can be used for thepurpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include, but are not limited to, lactose and cornstarch. Lubricating agents, such as, but not limited to, magnesiumstearate, also are typically added. For oral administration in a capsuleform, useful diluents include, but are not limited to, lactose and driedcorn starch. When aqueous suspensions or emulsions are administeredorally, the active ingredient can be suspended or dissolved in an oilyphase combined with emulsifying or suspending agents. If desired,certain sweetening, flavoring, or coloring agents can be added.

Pharmaceutical compositions for topical administration according to thedescribed invention can be formulated as solutions, ointments, creams,suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils.Alternatively, topical formulations can be in the form of patches ordressings impregnated with active ingredient(s), which can optionallycomprise one or more excipients or diluents. In some preferredembodiments, the topical formulations include a material that wouldenhance absorption or penetration of the active agent(s) through theskin or other affected areas. The topical composition is useful fortreating disorders in the skin, such as melanoma and certaininflammatory disorders.

A topical composition contains a safe and effective amount of adermatologically acceptable carrier suitable for application to theskin. A “cosmetically acceptable” or “dermatologically-acceptable”composition or component refers a composition or component that issuitable for use in contact with human skin without undue toxicity,incompatibility, instability, allergic response, and the like. Thecarrier enables an active agent and optional component to be deliveredto the skin at an appropriate concentration(s). The carrier thus can actas a diluent, dispersant, solvent, or the like to ensure that the activematerials are applied to and distributed evenly over the selected targetat an appropriate concentration. The carrier can be solid, semi-solid,or liquid. The carrier can be in the form of a lotion, a cream, or agel, in particular one that has a sufficient thickness or yield point toprevent the active materials from sedimenting. The carrier can be inertor possess dermatological benefits. It also should be physically andchemically compatible with the active components described herein, andshould not unduly impair stability, efficacy, or other use benefitsassociated with the composition.

Treatment Methods

The described invention provides methods for treating in a subject anangiogenic disorder or a disorder of angiogenesis.

The terms “angiogenic disorder,” “disorder of angiogenesis,” and“angiogenesis disorder” are used interchangeably herein, and refer to adisorder characterized by pathological angiogenesis. A disordercharacterized by pathological angiogenesis refers to a disorder whereabnormal or aberrant angiogenesis, alone or in combination with others,contributes to causation, origination, or symptom of the disorder.Examples of this disorder include various cancers (e.g., vascularizedtumors), eye disorders, inflammatory disorders, and others.

Typical vascularized tumors that can be treated with the method includesolid tumors, particularly carcinomas, which require a vascularcomponent for the provision of oxygen and nutrients. Exemplary solidtumors include, but are not limited to, carcinomas of the lung, breast,bone, ovary, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate, thyroid, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,glioblastomas, neuroblastomas, Kaposi's sarcoma, and sarcomas.

A number of disorders or conditions, other than cancer, also can betreated with the above-described method. Examples include arthritis,rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy,age-related macular degeneration, Grave's disease, vascular restenosis(including restenosis following angioplasty), arteriovenousmalformations (AVM), meningioma, hemangioma, neovascular glaucoma,chronic kidney disease, diabetic nephropathy, polycystic kidney disease,interstitial lung disease, pulmonary hypertension, chronic obstructivepulmonary disease (COPD), emphysema, autoimmune hepatitis, chronicinflammatory liver disease, hepatic cirrhosis, cutaneous T-celllymphoma, rosacea, and basal cell carcinoma.

Other treatment targets include those described in, e.g., USApplications 2009004297, 20090175791, and 20070161553, such asangiofibroma, atherosclerotic plaques, corneal graft neovascularization,hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenicgranuloma retrolental fibroplasia, scleroderma, trachoma, vascularadhesions, synovitis, dermatitis, various other inflammatory diseasesand disorders, and endometriosis.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human mammals, non-human primates (particularly higher primates),dog, rodent (e.g., mouse or rat), guinea pig, cat, and rabbit, andnon-mammals, such as birds, amphibians, reptiles, etc. In oneembodiment, the subject is a human. In another embodiment, the subjectis an experimental animal or animal suitable as a disease model. Asubject to be treated for a disorder can be identified by standarddiagnosing techniques for the disorder.

Optionally, the subject can be examined for mutation, expression level,or activity level of one or more of the genes or proteins mentionedabove by methods known in the art or described above before treatment.If the subject has a particular mutation in the gene, or if the geneexpression or activity level is, for example, greater in a sample fromthe subject than that in a sample from a normal person, the subject is acandidate for treatment.

To confirm the inhibition or treatment, one can evaluate and/or verifythe inhibition of endothelial recruitment or resulting angiogenesisusing technology known in the art before and/or after the administeringstep. Exemplary technologies include angiography or arteriography, amedical imaging technique used to visualize the inside, or lumen, ofblood vessels and organs of the body, can generally be done by injectinga radio-opaque contrast agent into the blood vessel and imaging usingX-ray based techniques such as fluoroscopy.

“Treating” or “treatment” refers to administration of a compound oragent to a subject who has a disorder with the purpose to cure,alleviate, relieve, remedy, delay the onset of, prevent, or amelioratethe disorder, the symptom of the disorder, the disease state secondaryto the disorder, or the predisposition toward the disorder.

An “effective amount” or “therapeutically effective amount” refers to anamount of the compound or agent that is capable of producing a medicallydesirable result in a treated subject. The treatment method can beperformed in vivo or ex vivo, alone or in conjunction with other drugsor therapy. A therapeutically effective amount can be administered inone or more administrations, applications or dosages and is not intendedto be limited to a particular formulation or administration route.

The agent can be administered in vivo or ex vivo, alone orco-administered in conjunction with other drugs or therapy, i.e., acocktail therapy. As used herein, the term “co-administration” or“co-administered” refers to the administration of at least two agent(s)or therapies to a subject. For example, in the treatment of tumors,particularly vascularized, malignant tumors, the agents can be usedalone or in combination with, e.g., chemotherapeutic, radiotherapeutic,apoptopic, anti-angiogenic agents and/or immunotoxins or coaguligands.

In some embodiments, the co-administration of two or moreagents/therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents/therapies used may vary.

In an in vivo approach, a compound or agent is administered to asubject. Generally, the compound is suspended in apharmaceutically-acceptable carrier (such as, for example, but notlimited to, physiological saline) and administered orally or byintravenous infusion, or injected or implanted subcutaneously,intramuscularly, intrathecally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of compounds available and the different efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by i.v.injection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the compound in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) can increase theefficiency of delivery, particularly for oral delivery.

Diagnosis

The described invention also provides diagnosis kits and methods. Asubject having cancer cells or a cells prone to tumorigenesis can bediagnosed based on the expression or activity of one or more of theabove-described genes or polypeptides in a test sample from the subject.The polypeptide and nucleic acids can be used as markers to indicate thepresence or absence of a cancer cell or cell prone to tumorigenesis.Diagnostic and prognostic assays of the described invention includemethods for assessing the expression level of the polypeptide or nucleicacid.

The presence, level, or absence of the polypeptide or nucleic acid in atest sample can be evaluated by obtaining a test sample from a testsubject and contacting the test sample with a compound or an agentcapable of detecting the polypeptide or nucleic acid (e.g., mRNA probe,genomic cDNA probe, or cDNA probe). The “test sample” can includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. The level ofexpression of the gene can be measured in a number of ways, including,but not limited to, measuring the mRNA encoded by the gene; measuringthe amount of polypeptide encoded by the gene; or measuring the activityof polypeptide encoded by the gene.

The level of mRNA corresponding to the gene in a cell can be determinedboth by in situ and by in vitro formats. Messenger RNA isolated from atest sample can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses, PCRanalyses, and probe arrays. For example, one diagnostic method for thedetection of mRNA levels involves contacting the isolated mRNA with anucleic acid probe that can hybridize to the mRNA encoded by the gene.The probe can be a full-length nucleic acid, or a portion thereof, suchas an oligonucleotide of at least 10 nucleotides in length andsufficient to specifically hybridize under stringent conditions to mRNAor genomic DNA.

In one format, mRNA (or cDNA prepared from it) is immobilized on asurface and contacted with the probes, for example, by running theisolated mRNA on an agarose gel and transferring the mRNA from the gelto a membrane, such as nitrocellulose. In another format, the probes areimmobilized on a surface and the mRNA (or cDNA) is contacted with theprobes, for example, in a gene chip array. A skilled artisan can adaptknown mRNA detection methods for detecting the level of mRNA.

The level of mRNA (or cDNA prepared from it) in a sample encoded by oneor more of the above-mentioned genes can be evaluated with nucleic acidamplification, e.g., by standard PCR (U.S. Pat. No. 4,683,202), RT-PCR(Bustin S. J Mol. Endocrinol. 25:169-93, 2000), quantitative PCR (Ong Y.et al., Hematology. 7:59-67, 2002), real time PCR (Ginzinger D. ExpHematol. 30:503-12, 2002), and in situ PCR (Thaker V. Methods Mol. Biol.115:379-402, 1999), or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniquesknown in the art. As used herein, “amplification primers” are defined asbeing a pair of nucleic acid molecules that can anneal to 5′ or 3′regions of a gene (plus and minus strands, respectively, or vice-versa)and contain a short region in between. Under appropriate conditions andwith appropriate reagents, such primers permit the amplification of anucleic acid molecule having the nucleotide sequence flanked by theprimers.

For in situ methods, a cell or tissue sample can be prepared andimmobilized on a support, such as, but not limited to, a glass slide,and then contacted with a probe that can hybridize to genomic DNA onchromosomes or mRNA that encodes the corresponding polypeptide.

In another embodiment, the methods of the described invention furtherinclude contacting a control sample with a compound or agent capable ofdetecting mRNA, or genomic DNA, and comparing the presence of mRNA orgenomic DNA in the control sample with the presence of mRNA or genomicDNA in the test sample.

The above-described nucleic acid-based diagnostic methods can providequalitative and quantitative information to determine whether a subjecthas or is predisposed to a disease associated with aberrant geneexpression and aberrant angiogenesis, e.g., cancers.

A variety of methods can be used to determine the level of one or moreof the above-mentioned polypeptide. In general, these methods includecontacting an agent that selectively binds to the polypeptide, such asan antibody, to evaluate the level of polypeptide in a sample.Antibodies can be polyclonal, or monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) also can be used. In anotherembodiment, the antibody bears a detectable label. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by physically linking a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with a detectable substance. Forexample, an antibody with a rabbit Fc region can be indirectly labeledusing a second antibody directed against the rabbit Fc region, whereinthe second antibody is coupled to a detectable substance. Examples ofdetectable substances are provided herein. Appropriate detectablesubstance or labels include, but are not limited to, radio isotopes (forexample, but not limited to, ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (forexample, but not limited to, alkaline phosphatase, horseradishperoxidase, luciferase, or β-glactosidase), fluorescent moieties orproteins (for example, but not limited to, fluorescein, rhodamine,phycoerythrin, GFP, or BFP), or luminescent moieties (for example, butnot limited to, Qdot™ nanoparticles by the Quantum Dot Corporation, PaloAlto, Calif.).

The detection methods can be used to detect one or more of theabove-mentioned polypeptide in a biological sample in vitro as well asin vivo. In vitro techniques for detection of the polypeptide includeELISAs, immuno-precipitations, immunofluorescence, EIA, RIA, and Westernblotting analysis. In vivo techniques for detection of the polypeptideinclude introducing into a subject a labeled antibody. For example, theantibody can be labeled with a detectable substance as described above.The presence and location of the detectable substance in a subject canbe detected by standard imaging techniques.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated with aberrantexpression or activity of one or more of the above-mentionedpolypeptides. As described herein, examples of such a disease ordisorder include those described above.

The prognostic assays described herein can be used to determine whethera subject is suitable to be administered with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disorder, such ascancer. For example, such assays can be used to determine whether asubject can be administered with a cytotoxic drug to treat the disorder.

Information obtained from practice of the above assays is useful inprognostication, identifying progression of, and clinical management ofdiseases and other deleterious conditions affecting an individual'shealth status. In some embodiments, the foregoing diagnostic assaysprovide information useful in prognostication, identifying progressionof and management of malignancies (cancers) that are characterized byabnormal, pathological angiogenesis. The information more specificallyassists the clinician in designing chemotherapeutic or other treatmentregimes to eradicate such malignancies from the body of an afflictedmammal, e.g., a human.

Example 1 Methods and Materials

This example describes general methods and materials used in Examples2-8.

Cell Culture

All cell lines were propagated as described in Tavazoie, S. F. et al.,Nature 451 (7175), 147 (2008). 293T cells were cultured with DMEM mediasupplemented with 10% FBS; H29 cells were cultured with DMEM mediasupplemented with 10% FBS, 20 ng/mL doxycycline, 2 μg/mL puromycin, and0.3 mg/mL G418; and HUVEC cells were cultured with EGM-2 media (CC-3162,Lonza, Basel, Switzerland). The MDA-MB-231 and CN34 breast cancer cellline and its metastatic derivatives LM2, BoM2 and Lm1a are described inMinn, A. J. et al., Nature 436 (7050), 518 (2005).

Animal Studies

All animal work was conducted in accordance with a protocol approved bythe Institutional Animal Care and Use Committee (IACUC) at TheRockefeller University. Age-matched female NOD/SCID mice (6-8 week old)were used for both orthotopic mammary fat pad tumor initiation assays(Minn, A. J. et al., Nature 436 (7050), 518 (2005) and for lungmetastasis assays (Tavazoie, S. F. et al., Nature 451 (7175), 147(2008)). Eight-week old age-matched female athymic mice were used forsystemic metastasis assays (Kang, Y. et al., Cancer Cell 3 (6), 537(2003) and Yin, J. J. et al., J Clin Invest 103 (2), 197 (1999)).

Inducible miR-126 expression was obtained by cloning pre-miR-126 intothe tet-ON containing pTripz vector (Thermo Scientific, Huntsville,Ala.). At day 3, 2 mg/ml doxycycline (Sigma Aldrich) was added to thedrinking water containing 5% sucrose. Control mice were given drinkingwater with 5% sucrose.

Generation of Lentivirus, Retrovirus, Knockdown and Over-ExpressingCells

For generation of lentivirus, 1×10⁶ 293T cells were seeded onto a 10 cmplate and incubated for 24 h. Twelve micrograms of vector K (Gag/Pol), 6μg of vector A (Env) and 12 μg of the appropriate shRNA plasmid werethen co-transfected into the 293T cells using 40 μL, of TRANSIT®-293transfection reagent (MIR 2700, MIRUS BIO LLC, Madison, Wis.). After 16h, the media was replaced with fresh antibiotic-free DMEM supplementedwith 10% FBS. After another 24 h, the virus was harvested by spinningfor min at 1,500 g before being filtered through a 0.45 μm filter. Forgeneration of retrovirus, H29 cells were seeded onto a 10-cm plate andallowed to grow to 90% confluence. Ten micrograms of the appropriateplasmid was then transfected into H29 cells using 60 μl ofLIPOFECTAMINE™ 2000 transfection reagent (11668-019, INVITROGEN by LIFETECHNOLOGIES, Carlsbad, Calif.). After 16 h, the media were replacedwith fresh antibiotic-free DMEM supplemented with 10% FBS. After another48 h, the virus was harvested by spinning for 5 min at 1,500 g andfiltered through a 0.45 μm filter. Two milliliters of the appropriatevirus was used to transduce 50K cancer cells in the presence of 10 μg/mLof polybrene (TR-1003-G, MILLIPORE, Billerica, A). After 24 h, the mediawas changed to DMEM supplemented with 10% FBS and 2 μg/mL puromycin(lentivirus) or 10 μg/mL blasticidin for selection. After another 72 h,the cells were washed and allowed to grow in D10F and tested for knockdown of the gene of interest by qPCR.

Endothelial Recruitment

Cancer cells (25,000) were seeded into 24-well plates approximately 24 hbefore the start of the recruitment assay. HUVEC cells were serumstarved in EGM-2 media supplemented with 0.2% FBS for 24 hours. TheHUVEC cells were then labeled with CELLTRACKER Red CMTPX dye (C34552,INVITROGEN) for 45 min and rescued in EGM-2 media supplemented with 2%FBS for 30 min. Meanwhile, cancer cells were washed with PBS and 1 mL0.2% FBS EGM-2 media was added to each well. Each well was then fittedwith a 3.0 μm HTS FLUROBLOCK Insert (351151, BD FALCON, San Jose,Calif.). For antibody experiments, the appropriate concentration of eachantibody was then added to each well: 50 ng/mL anti-IGFBP2 (AF674, R&DSYSTEMS, Minneapolis, Minn.), 20 μg/mL anti-IGF-1 (AF-291-NA, R&DSYSTEMS), 40 μg/mL anti-IGF-2 (MAB292, R&D SYSTEMS), 20 μg/mL anti-IGF1R(MAB391, R&D SYSTEMS), 5 μg/mL anti-IGF2R (AF2447, R&D Systems) andanti-IgG (AB-108-C, R&D SYSTEMS). For endothelial recruitment assaysthat require pre-incubation with antibodies, either HUVEC cells orcancer cells were then incubated with 20 μg/mL anti-IGF1R or control IgGantibody for 1 h and washed once with PBS. The HUVEC cells were thenserum starved for 1 h before being resuspended 0.2% FBS EGM-2 at 100KHUVECs per mL.

The resuspension (0.5 mL) was then added into each FLUOROBLOCK insertand the recruitment assay was allowed to proceed for 16 h. Aftercompletion of the assay, FLUOROBLOCK inserts were fixed with 4%paraformaldehyde for 15 min and mounted onto slides with VECTASHIELDmounting media (H-1000, VECTOR LABORATORIES, Burlingame, Calif.). Threeimages of each insert were taken and the images were analyzed usingIMAGEJ (NIH).

Chemotaxis Assay

Matrigel (250 μl, BD BIOSCIENCES, #356231) containing given amounts ofbovine serum albumin (A2153, Sigma Aldrich), rhIGFBP2 (674-B2, R&DSystems), rhGas6 (885-GS, R&D Systems), anti-IGF1R (MAB391, R&D Systems)and MerFc (891-MR-100, R&D Systems) were allowed to solidify at thebottom of a 24 well plate for 30 min before 250 μl HUVEC mediacontaining 0.2% FBS were added. A 3.0-μm HTS Fluoroblock Insert (351151,BD Falcon) was then placed in each well. HUVEC cells were labeled withCellTracker Red CMTPX dye (C34552, Invitrogen) before resuspending 300KHUVECs per mL of 0.2% FBS EGM-2. 0.5 mL of the resuspension was thenadded into each Fluoroblock insert and the assay allowed proceeding for20 h. The inserts were then fixed for 15 min in 4% paraformaldehyde andmounted onto slides with VectaShield mounting media (H-1000, VectorLaboratories). 5 fields of the basal side of each insert were thenimaged and the images were analyzed using ImageJ (NIH).

Migration Assay

HUVEC cells were grown to 90% confluence and stimulated in the givenconcentrations of bovine serum albumin (Sigma Aldrich, #A2153), rhIGFBP2(674-B2, R&D Systems) and anti-IGF1R (MAB391, R&D Systems) in HUVECmedia containing 0.2% FBS for 40 min at 37° C. The cells were thentrypsinized and 50K cells were added into HTS Fluoroblock Inserts(351151, BD Falcon). After 24 hours in 37° C. with 5% CO₂, the insertswere removed, the membrane excised and fixed in 4% paraformaldehyde.HUVEC cells that had migrated to the basal side of the membrane werevisualized with DAPI and counted in 5 fields per membrane using Image J(NIH).

Endothelial Adhesion

HUVEC cells were seeded on a 6-cm plate and allowed to grow toconfluence. Cancer cells were serum starved in DMEM media supplementedwith 0.2% FBS for 30 min, labeled with CELLTRACKER Green CMFDA dye(C7025, Invitrogen) for 45 min and incubated in DMEM media supplementedwith 10% FBS for 30 min. Cancer cells were then trypsinized andresuspended in 10% FBS/DMEM to 10K cells/mL. Five milliliters of theresuspension was then added to each plate of HUVECs and the plate wasincubated at 37° C. for 10 min. The plates were then washed gently withPBS and fixed with 4% paraformaldehyde for 15 min. Each plate was thentreated with 1 mL of PBS and 6 images were taken from each plate. Thenumber of cancer cells adherent to the HUVEC cells were then quantifiedusing IMAGEJ.

Endothelial Proliferation

Cancer cells (1×10⁶) were seeded to a 10-cm plate and allowed to growfor 24 h. The cancer cells were then washed gently with PBS and EGM-2media supplemented with 2% FBS was added to each plate. The conditionedEGM-2 media was collected after 24 h. HUVEC cells (25K) were seeded intriplicate in a 6-well plate and allowed to grow for 16 h. The HUVECcells were then washed gently with PBS and 3 mL conditioned EGM-2 mediawas added to each well. After 48 h, the conditioned media was replacedwith another 3 mL of conditioned media. After another 48 h, the cellswere trypsinized and counted using a haemocytometer.

Tube Formation Assay

Tube formation assay was performed according to manufacturer's protocol(354149, BD BIOCOAT™ ANGIOGENESIS SYSTEM—Endothelial Cell TubeFormation). Briefly, HUVEC cells were serum starved in EGM-2 mediasupplemented with 0.2% FBS for 24 hours. The HUVEC cells were thenlabeled with CELLTRACKER Red CMTPX dye (C34552, INVITROGEN) for 45 minand subsequently treated in EGM-2 media supplemented with 2% FBS for 30min. Meanwhile, the tube formation assay plate, which was in 96-wellformat, was incubated at 37° C. for 30 min. The cancer cells and HUVECcells were trypsinized and resuspended at 400K/mL and 800K/mLrespectively in EGM-2 media supplemented with 2% FBS. The cancer celland HUVEC cell suspensions were then mixed at a 1:1 ratio and 50 μl ofeach mixture was seeded into each well of the tube formation assayplate. The assay plate was incubated at 37° C. for 16 h. Images of eachwell were taken and the images were processed using METAMORPH analysissoftware (MOLECULAR DEVICES, Inc.) to obtain the number of branch pointsper image.

Analysis of miRNA and mRNA Expression

Total RNA was extracted from various cell lines using the MIRVANA kit(AM1560, APPLIED BIOSYSTEMS, Austin, Tex.). TAQMAN microRNA assay(4427975-0002228, APPLIED BIOSYSTEMS) was used to quantify expressionlevels of mature miRNA as described in Tavazoie, S. F. et al., Nature451 (7175), 147 (2008). For quantification of mRNA, 400 ng of total RNAwere reverse transcribed using the cDNA First-Strand Synthesis Kit(18080-051, INVITROGEN). Approximately 4 ng of the resulting cDNA wasthen mixed with SYBR green PCR MASTER MIX (4309155, APPLIED BIOSYSTEMS)and appropriate primers (Table 1). Quantitative mRNA expression data wasobtained using an ABI PRISM 7900HT Real-Time PCR System (APPLIEDBIOSYSTEMS). Smad4 was used as an endogenous control for normalization.Expression analysis of human breast cancers at various disease stageswas performed using the TISSUESCAN qPCR Array Breast Cancer Panels 2 and3 (BCRT102 & BCRT103, ORIGENE, Rockville, Md.).

TABLE 1 SYBR green qPCR primers Gene Forward Reverse ABCB9GACCTTCACCTACCGCACTC CACAGGAGCTCTTCCCACTG BEX2 GCCCCGAAAGTAGGAAGCCTCCATTACTCCTGGGCCTAT BGLAP GGCGCTACCTGTATCAATGG TCAGCCAACTCGTCACAGTCCA12 CCAAGGCTACAATCTGTCTGC GGGCAGGTTCAGCTTCACT GDF15 CCGGATACTCACGCCAGAAGAGATACGCAGGTGCAGGT GEM GACAGCATGGACAGCGACT AACCATCAGGGTTCGTTCAT IGFBP2CCAAGAAGCTGCGACCAC GGGATGTGCAGGGAGTAGAG ITGB4 TCAGCCTCTCTGGGACCTTTATCCACACGGACACACTCC KIAA0746 GTTGTCTGTGCAGATGTACGC TAGCAGGGCCAGGTTAAAAAKLF4 GCCGCTCCATTACCAAGA TCTTCCCCTCTTTGGCTTG MARS AACAACCTGGGCAACTTCATACCATCTCAGGCACATAGCC MERTK GGAGACAGGACCAAAGC GGGCAATATCCACCATGAAC PADI4AAGTGCAAGCTGACCATCTG GCCGATCTCCATTTCATCC PHGDH TGGTGGAAAAGCAGAACCTTAACAATAAGGCCTTCACAGTCC PITPNC1 GCGCTACTACAAAGAATCTGAGGGAGCACATGATAGGCTGATGAC PSAT1 TCTTGTGCGGGAATTGCTA AAGGGGACAGCACTGAACTGPYGB TCCAGGGTCCTGTATCCAAA CCACGAAGTACTCCTGCTTCA RGC32TGCTGATCTTGACAAAACTTTAGC GCAGGTCCTCGGAACTTTCT SHMT2 GAGGGAGAAGGACAGGCAGTCTCGGCTGCAGAAGTTCTCT SMAD4 TGGCCCAGGATCAGTAGGT CATCAACACCAATTCCAGCA THBDAATTGGGAGCTTGGGAATG TGAGGACCTGATTAAGGCTAGG TNFSF4GTATCCTCGAATTCAAAGTATCAAAGT CTGAGTTGTTCTGCACCTTCA VIPR1CTGTCCCCTCATCTTCAAGC CAGCTGCGGCTTACATTG

miR-126 Target Prediction

Potential miR-126 targets were identified by using 3 sets of microarrayprofiles: LM2 control cells relative to LM2 cells over-expressingmiR-126 (GSE No. 23905) and 2 replicate arrays of MDA and LM2 cells (GSENo. 23904 and Minn, A. J. et al., Nature 436 (7050), 518 (2005). Withthese arrays, the following criteria were used to identify possiblemiR-126 targets genes: (1) Genes down-regulated more than 1.6 fold uponmiR-126 over-expression in LM2 cells and (2) Genes up-regulated by morethan 1.4 fold in one of the two LM2 versus MDA arrays. All potentialtargets were subsequently verified by qPCR.

Luciferase Reporter Assay

Luciferase reporter assay was performed as described in Tavazoie, S. F.et al., Nature 451 (7175), 147 (2008). Briefly the full-length 3′UTR'sand CDS's of ABCB9, IGFBP2, MERTK, PITPNC1, PSAT1, PYGB, SHMT2 and VIPR1were cloned into the psiCheck2 dual luciferase reporter vector (C8021,PROMEGA, Madison, Wis.). Listed below are the sequences of the CDS's and3′UTR's.

ABCB9 CDSATGCGGCTGTGGAAGGCGGTGGTGGTGACTTTGGCCTTCATGAGTGTGGACATCTGCGTGACCACGGCCATCTATGTCTTCAGCCACCTGGACCGCAGCCTCCTGGAGGACATCCGCCACTTCAACATCTTTGACTCGGTGCTGGATCTCTGGGCAGCCTGCCTGTACCGCAGCTGCCTGCTGCTGGGAGCCACCATTGGTGTGGCCAAGAACAGTGCGCTGGGGCCCCGGCGGCTGCGGGCCTCGTGGCTGGTCATCACCCTCGTGTGCCTCTTCGTGGGCATCTATGCCATGGTGAAGCTGCTGCTCTTCTCAGAGGTGCGCAGGCCCATCCGGGACCCCTGGTTTTGGGCCCTGTTCGTGTGGACGTACATTTCACTCGGCGCATCCTTCCTGCTCTGGTGGCTGCTGTCCACCGTGCGGCCAGGCACCCAGGCCCTGGAGCCAGGGGCGGCCACCGAGGCTGAGGGCTTCCCTGGGAGCGGCCGGCCACCGCCCGAGCAGGCGTCTGGGGCCACGCTGCAGAAGCTGCTCTCCTACACCAAGCCCGACGTGGCCTTCCTCGTGGCCGCCTCCTTCTTCCTCATCGTGGCAGCTCTGGGAGAGACCTTCCTGCCCTACTACACGGGCCGCGCCATTGATGGCATCGTCATCCAGAAAAGCATGGATCAGTTCAGCACGGCTGTCGTCATCGTGTGCCTGCTGGCCATTGGCAGCTCATTTGCCGCAGGTATTCGGGGCGGCATTTTTACCCTCATATTTGCCAGACTGAACATTCGCCTTCGAAACTGTCTCTTCCGCTCACTGGTGTCCCAGGAGACAAGCTTCTTTGATGAGAACCGCACAGGGGACCTCATCTCCCGCCTGACCTCGGACACCACCATGGTCAGCGACCTGGTCTCCCAGAACATCAATGTCTTCCTGCGGAACACAGTCAAGGTCACGGGCGTGGTGGTCTTCATGTTCAGCCTCTCATGGCAGCTCTCCTTGGTCACCTTCATGGGCTTCCCCATCATCATGATGGTGTCCAACATCTACGGCAAGTACTACAAGAGGCTCTCCAAAGAGGTCCAGAATGCCCTGGCCAGAGCGAGCAACACGGCGGAGGAGACCATCAGTGCCATGAAGACTGTCCGGAGCTTCGCCAATGAGGAGGAGGAGGCAGAGGTGTACCTGCGGAAGCTGCAGCAGGTGTACAAGCTGAACAGGAAGGAGGCAGCTGCCTACATGTACTACGTCTGGGGCAGCGGGCTCACACTGCTGGTGGTCCAGGTCAGCATCCTCTACTACGGGGGCCACCTTGTCATCTCAGGCCAGATGACCAGCGGCAACCTCATCGCCTTCATCATCTACGAGTTTGTCCTGGGAGATTGTATGGAGTCCGTGGGCTCCGTCTACAGTGGCCTGATGCAGGGAGTGGGGGCTGCTGAGAAGGTGTTCGAGTTCATCGACCGGCAGCCGACCATGGTGCACGATGGCAGCTTGGCCCCCGACCACCTGGAGGGCCGGGTGGACTTTGAGAATGTGACCTTCACCTACCGCACTCGGCCCCACACCCAGGTCCTGCAGAATGTCTCCTTCAGCCTGTCCCCCGGCAAGGTGACGGCCCTGGTGGGGCCCTCGGGCAGTGGGAAGAGCTCCTGTGTCAACATCCTGGAGAACTTCTACCCCCTGGAGGGGGGCCGGGTGCTGCTGGACGGCAAGCCCATCAGCGCCTACGACCACAAGTACTTGCACCGTGTGATCTCCCTGGTGAGCCAGGAGCCCGTGCTGTTCGCCCGCTCCATCACGGATAACATCTCCTACGGCCTGCCCACTGTGCCTTTCGAGATGGTGGTGGAGGCCGCACAGAAGGCCAATGCCCACGGCTTCATCATGGAACTCCAGGACGGCTACAGCACAGAGACAGGGGAGAAGGGCGCCCAGCTGTCAGGTGGCCAGAAGCAGCGGGTGGCCATGGCCCGGGCTCTGGTGCGGAACCCCCCAGTCCTCATCCTGGATGAAGCCACCAGCGCTTTGGATGCCGAGAGCGAGTATCTGATCCAGCAGGCCATCCATGGCAACCTGCAGAAGCACACGGTACTCATCATCGCGCACCGGCTGAGCACCGTGGAGCACGCGCACCTCATTGTGGTGCTGGACAAGGGCCGCGTAGTGCAGCAGGGCACCCACCAGCAGCTGCTGGCCCAGGGCGGCCTCTACGCCAAGCTGGTGCAGCGGCAGATGCTGGGGCTTCAGCCCGCCGCAGACTTCACAGCTGGCCACAACGAGCCTGTAGCCAACGGCAGTCACAAGGCCTGA ABCB9 3′UTRTGGGGGGCCCCTGCTTCTCCCGGTGGGGCAGAGGACCCGGTGCCTGCCTGGCAGATGTGCCCACGGAGGCCCCCAGCTGCCCTCCGAGCCCAGGCCTGCAGCACTGAAAGACGACCTGCCATGTCCCATGGATCACCGCTTCCTGCATCTTGCCCCTGGTCCCTGCCCCATTCCCAGGGCACTCCTTACCCCTGCTGCCCTGAGCCAACGCCTTCACGGACCTCCCTAGCCTCCTAAGCAAAGGTAGAGCTGCCTTTTTAAACCTAGGTCTTACCAGGGTTTTTACTGTTTGGTTTGAGGCACCCCAGTCAACTCCTAGATTTCAAAAACCTTTTTCTAATTGGGAGTAATGGCGGGCACTTTCACCAAGATGTTCTAGAAACTTCTGAGCCAGGAGTGAATGGCCCTTCCTTAGTAGCCTGGGGGATGTCCAGAGACTAGGCCTCTCCCCTTTACCCCTCCAGAGAAGGGGCTTCCCTGTCCCGGAGGGAGACACGGGGAACGGGATTTTCCGTCTCTCCCTCTTGCCAGCTCTGTGAGTCTGGCCAGGGCGGGTAGGGAGCGTGGAGGGCATCTGTCTGCCATCGCCCGCTGCCAATCTAAGCCAGTCTCACTGTGAACCACACGAAACCTCAACTGGGGGAGTGAGGGGCTGGCCAGGTCTGGAGGGGCCTCAGGGGTGCCCCCAGCCCGGCACCCAGCGCTTTCGCCCCTCGTCCACCCACCCCTGGCTGGCAGCCTCCCTCCCCACACCCGCCCCTGTGCTCTGCTGTCTGGAGGCCACGTGGATGTTCATGAGATGCATTCTCTTCTGTCTTTGGTGGATGGGATGGTGGCAAAGCCCAGGATCTGGCTTTGCCAGAGGTTGCAACATGTTGAGAGAACCCGGTCAATAAAGTGTACTACCTCTTACCCCTAA IGFBP2 CDSATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCCGCCGCCGCCGCTGCTGCCGCTGCTGCTGCTGCTACTGGGCGCGAGTGGCGGCGGCGGCGGGGCGCGCGCGGAGGTGCTGTTCCGCTGCCCGCCCTGCACACCCGAGCGCCTGGCCGCCTGCGGGCCCCCGCCGGTTGCGCCGCCCGCCGCGGTGGCCGCAGTGGCCGGAGGCGCCCGCATGCCATGCGCGGAGCTCGTCCGGGAGCCGGGCTGCGGCTGCTGCTCGGTGTGCGCCCGGCTGGAGGGCGAGGCGTGCGGCGTCTACACCCCGCGCTGCGGCCAGGGGCTGCGCTGCTATCCCCACCCGGGCTCCGAGCTGCCCCTGCAGGCGCTGGTCATGGGCGAGGGCACTTGTGAGAAGCGCCGGGACGCCGAGTATGGCGCCAGCCCGGAGCAGGTTGCAGACAATGGCGATGACCACTCAGAAGGAGGCCTGGTGGAGAACCACGTGGACAGCACCATGAACATGTTGGGCGGGGGAGGCAGTGCTGGCCGGAAGCCCCTCAAGTCGGGTATGAAGGAGCTGGCCGTGTTCCGGGAGAAGGTCACTGAGCAGCACCGGCAGATGGGCAAGGGTGGCAAGCATCACCTTGGCCTGGAGGAGCCCAAGAAGCTGCGACCACCCCCTGCCAGGACTCCCTGCCAACAGGAACTGGACCAGGTCCTGGAGCGGATCTCCACCATGCGCCTTCCGGATGAGCGGGGCCCTCTGGAGCACCTCTACTCCCTGCACATCCCCAACTGTGACAAGCATGGCCTGTACAACCTCAAACAGTGCAAGATGTCTCTGAACGGGCAGCGTGGGGAGTGCTGGTGTGTGAACCCCAACACCGGGAAGCTGATCCAGGGAGCCCCCACCATCCGGGGGGACCCCGAGTGTCATCTCTTCTACAATGAGCAGCAGGAGGCTCGCGGGGTGCACACCCAGCGGATGCAGTAG IGFBP2 3′UTRACCGCAGCCAGCCGGTGCCTGGCGCCCCTGCCCCCCGCCCCTCTCCAAACACCGGCAGAAAACGGAGAGTGCTTGGGTGGTGGGTGCTGGAGGATTTTCCAGTTCTGACACACGTATTTATATTTGGAAAGAGACCAGCACCGAGCTCGGCACCTCCCCGGCCTCTCTCTTCCCAGCTGCAGATGCCACACCTGCTCCTTCTTGCTTTCCCCGGGGGAGGAAGGGGGTTGTGGTCGGGGAGCTGGGGTACAGGTTTGGGGAGGGGGAAGAGAAATTTTTATTTTTGAACCCCTGTGTCCCTTTTGCATAAGATTAAAGGAAGGAAAAGTA AMERTK CDSATGGGGCCGGCCCCGCTGCCGCTGCTGCTGGGCCTCTTCCTCCCCGCGCTCTGGCGTAGAGCTATCACTGAGGCAAGGGAAGAAGCCAAGCCTTACCCGCTATTCCCGGGACCTTTTCCAGGGAGCCTGCAAACTGACCACACACCGCTGTTATCCCTTCCTCACGCCAGTGGGTACCAGCCTGCCTTGATGTTTTCACCAACCCAGCCTGGAAGACCACATACAGGAAACGTAGCCATTCCCCAGGTGACCTCTGTCGAATCAAAGCCCCTACCGCCTCTTGCCTTCAAACACACAGTTGGACACATAATACTTTCTGAACATAAAGGTGTCAAATTTAATTGCTCAATCAGTGTACCTAATATATACCAGGACACCACAATTTCTTGGTGGAAAGATGGGAAGGAATTGCTTGGGGCACATCATGCAATTACACAGTTTTATCCAGATGATGAAGTTACAGCAATAATCGCTTCCTTCAGCATAACCAGTGTGCAGCGTTCAGACAATGGGTCGTATATCTGTAAGATGAAAATAAACAATGAAGAGATCGTGTCTGATCCCATCTACATCGAAGTACAAGGACTTCCTCACTTTACTAAGCAGCCTGAGAGCATGAATGTCACCAGAAACACAGCCTTCAACCTCACCTGTCAGGCTGTGGGCCCGCCTGAGCCCGTCAACATTTTCTGGGTTCAAAACAGTAGCCGTGTTAACGAACAGCCTGAAAAATCCCCCTCCGTGCTAACTGTTCCAGGCCTGACGGAGATGGCGGTCTTCAGTTGTGAGGCCCACAATGACAAAGGGCTGACCGTGTCCAAGGGAGTGCAGATCAACATCAAAGCAATTCCCTCCCCACCAACTGAAGTCAGCATCCGTAACAGCACTGCACACAGCATTCTGATCTCCTGGGTTCCTGGTTTTGATGGATACTCCCCGTTCAGGAATTGCAGCATTCAGGTCAAGGAAGCTGATCCGCTGAGTAATGGCTCAGTCATGATTTTTAACACCTCTGCCTTACCACATCTGTACCAAATCAAGCAGCTGCAAGCCCTGGCTAATTACAGCATTGGTGTTTCCTGCATGAATGAAATAGGCTGGTCTGCAGTGAGCCCTTGGATTCTAGCCAGCACGACTGAAGGAGCCCCATCAGTAGCACCTTTAAATGTCACTGTGTTTCTGAATGAATCTAGTGATAATGTGGACATCAGATGGATGAAGCCTCCGACTAAGCAGCAGGATGGAGAACTGGTGGGCTACCGGATATCCCACGTGTGGCAGAGTGCAGGGATTTCCAAAGAGCTCTTGGAGGAAGTTGGCCAGAATGGCAGCCGAGCTCGGATCTCTGTTCAAGTCCACAATGCTACGTGCACAGTGAGGATTGCAGCCGTCACCAGAGGGGGAGTTGGGCCCTTCAGTGATCCAGTGAAAATATTTATCCCTGCACACGGTTGGGTAGATTATGCCCCCTCTTCAACTCCGGCGCCTGGCAACGCAGATCCTGTGCTCATCATCTTTGGCTGCTTTTGTGGATTTATTTTGATTGGGTTGATTTTATACATCTCCTTGGCCATCAGAAAAAGAGTCCAGGAGACAAAGTTTGGGAATGCATTCACAGAGGAGGATTCTGAATTAGTGGTGAATTATATAGCAAAGAAATCCTTCTGTCGGCGAGCCATTGAACTTACCTTACATAGCTTGGGAGTCAGTGAGGAACTACAAAATAAACTAGAAGATGTTGTGATTGACAGGAATCTTCTAATTCTTGGAAAAATTCTGGGTGAAGGAGAGTTTGGGTCTGTAATGGAAGGAAATCTTAAGCAGGAAGATGGGACCTCTCTGAAAGTGGCAGTGAAGACCATGAAGTTGGACAACTCTTCACAGCGGGAGATCGAGGAGTTTCTCAGTGAGGCAGCGTGCATGAAAGACTTCAGCCACCCAAATGTCATTCGACTTCTAGGTGTGTGTATAGAAATGAGCTCTCAAGGCATCCCAAAGCCCATGGTAATTTTACCCTTCATGAAATACGGGGACCTGCATACTTACTTACTTTATTCCCGATTGGAGACAGGACCAAAGCATATTCCTCTGCAGACACTATTGAAGTTCATGGTGGATATTGCCCTGGGAATGGAGTATCTGAGCAACAGGAATTTTCTTCATCGAGATTTAGCTGCTCGAAACTGCATGTTGCGAGATGACATGACTGTCTGTGTTGCGGACTTCGGCCTCTCTAAGAAGATTTACAGTGGCGATTATTACCGCCAAGGCCGCATTGCTAAGATGCCTGTTAAATGGATCGCCATAGAAAGTCTTGCAGACCGAGTCTACACAAGTAAAAGTGATGTGTGGGCATTTGGCGTGACCATGTGGGAAATAGCTACGCGGGGAATGACTCCCTATCCTGGGGTCCAGAACCATGAGATGTATGACTATCTTCTCCATGGCCACAGGTTGAAGCAGCCCGAAGACTGCCTGGATGAACTGTATGAAATAATGTACTCTTGCTGGAGAACCGATCCCTTAGACCGCCCCACCTTTTCAGTATTGAGGCTGCAGCTAGAAAAACTCTTAGAAAGTTTGCCTGACGTTCGGAACCAAGCAGACGTTATTTACGTCAATACACAGTTGCTGGAGAGCTCTGAGGGCCTGGCCCAGGGCTCCACCCTTGCTCCACTGGACTTGAACATCGACCCTGACTCTATAATTGCCTCCTGCACTCCCCGCGCTGCCATCAGTGTGGTCACAGCAGAAGTTCATGACAGCAAACCTCATGAAGGACGGTACATCCTGAATGGGGGCAGTGAGGAATGGGAAGATCTGACTTCTGCCCCCTCTGCTGCAGTCACAGCTGAAAAGAACAGTGTTTTACCGGGGGAGAGACTTGTTAGGAATGGGGTCTCCTGGTCCCATTCGAGCATGCTGCCCTTGGGAAGCTCATTGCCCGATGAACTTTTGTTTGCTGACGACTCCTCAGAAGGCTCAGAAGTCCTGATGTGA MERTK 3′UTRGGAGAGGTGCGGGGAGACATTCCAAAAATCAAGCCAATTCTTCTGCTGTAGGAGAATCCAATTGTACCTGATGTTTTTGGTATTTGTCTTCCTTACCAAGTGAACTCCATGGCCCCAAAGCACCAGATGAATGTTGTTAAGTAAGCTGTCATTAAAAATACATAATATATATTTATTTAAAGAGAAAAAATATGTGTATATCATGGAAAAAGACAAGGATATTTTAATAAAACATTACTTATTTCATTTCACTTATCTTGCATATCTTAAAATTAAGCTTCAGCTGCTCCTTGATATTAACATTTGTACAGAGTTGAAGTTGTTTTTTCAAGTTCTTTTCTTTTTCATGACTATTAAATGTAAAAATATTTGTAAAATGAAATGCCATATTTGACTTGGCTTCTGGTCTTGATGTATTTGATAAGAATGATTCATTCAATGTTTAAAGTTGTATAACTGATTAATTTTCTGATATGGCTTCCTAATAAAATATGAATAAGGAAG PITPNC1 isoform A CDS ATGCTGCTGA AAGAGTACCG GATCTGCATG CCGCTCACCG TAGACGAGTA CAAAATTGGA CAGCTGTACA TGATCAGCAA ACACAGCCAT GAACAGAGTG ACCGGGGAGA AGGGGTGGAG GTCGTCCAGA ATGAGCCCTT TGAGGACCCT CACCATGGCA ATGGGCAGTT CACCGAGAAG CGGGTGTATC TCAACAGCAA ACTGCCTAGT TGGGCTAGAG CTGTTGTCCC CAAAATATTT TATGTGACAG AGAAGGCTTG GAACTATTAT CCCTACACAA TTACAGAATA CACATGTTCC TTTCTGCCGA AATTCTCCAT TCATATAGAA ACCAAGTATG AGGACAACAA AGGAAGCAAT GACACCATTT TCGACAATGA AGCCAAAGAC GTGGAGAGAG AAGTTTGCTT TATTGATATT GCCTGCGATG AAATTCCAGA GCGCTACTAC AAAGAATCTG AGGATCCTAA GCACTTCAAG TCAGAGAAGA CAGGACGGGG ACAGTTGAGG GAAGGCTGGA GAGATAGTCA TCAGCCTATC ATGTGCTCCT ACAAGCTGGT GACTGTGAAG TTTGAGGTCT GGGGGCTTCA GACCAGAGTG GAACAATTTG TACACAAGGT GGTCCGAGAC ATTCTGCTGA TTGGACATAG ACAGGCTTTT GCATGGGTTG ATGAGTGGTA TGACATGACA ATGGATGAAG TCCGAGAATT TGAACGAGCC ACTCAGGAAG CCACCAACAA GAAAATCGGC ATTTTCCCAC CTGCAATTTC TATCTCCAGC ATCCCCCTGC TGCCTTCTTC CGTCCGCAGT GCGCCTTCTA GTGCTCCATC CACCCCTCTC TCCACAGACG CACCCGAATT TCTGTCCGTT CCCAAAGATC GGCCCCGGAA AAAGTCTGCC CCAGAAACTC TCACACTTCC AGACCCTGAG AAAAAAGCCA CCCTGAATTT ACCCGGCATG CACTCTTCAG ATAAGCCATG TCGGCCCAAA TCTGAGTAA PITPNC1 isoform B CDSATGCTGCTGAAAGAGTACCGGATCTGCATGCCGCTCACCGTAGACGAGTACAAAATTGGACAGCTGTACATGATCAGCAAACACAGCCATGAACAGAGTGACCGGGGAGAAGGGGTGGAGGTCGTCCAGAATGAGCCCTTTGAGGACCCTCACCATGGCAATGGGCAGTTCACCGAGAAGCGGGTGTATCTCAACAGCAAACTGCCTAGTTGGGCTAGAGCTGTTGTCCCCAAAATATTTTATGTGACAGAGAAGGCTTGGAACTATTATCCCTACACAATTACAGAATACACATGTTCCTTTCTGCCGAAATTCTCCATTCATATAGAAACCAAGTATGAGGACAACAAAGGAAGCAATGACACCATTTTCGACAATGAAGCCAAAGACGTGGAGAGAGAAGTTTGCTTTATTGATATTGCCTGCGATGAAATTCCAGAGCGCTACTACAAAGAATCTGAGGATCCTAAGCACTTCAAGTCAGAGAAGACAGGACGGGGACAGTTGAGGGAAGGCTGGAGAGATAGTCATCAGCCTATCATGTGCTCCTACAAGCTGGTGACTGTGAAGTTTGAGGTCTGGGGGCTTCAGACCAGAGTGGAACAATTTGTACACAAGGTGGTCCGAGACATTCTGCTGATTGGACATAGACAGGCTTTTGCATGGGTTGATGAGTGGTATGATATGACAATGGATGATGTTCGGGAATACGAGAAAAACATGCATGAACAAACCAACATAAAAGTTTGCAATCAGCATTCCTCCCCTGTGGATGACATAGAGAGTCATGCCCAAACAAGTACATGA PITPNC1 3′UTRCAATGGATGAAGTCCGAGAATTTGAACGAGCCACTCAGGAAGCCACCAACAAGAAAATCGGCATTTTCCCACCTGCAATTTCTATCTCCAGCATCCCCCTGCTGCCTTCTTCCGTCCGCAGTGCGCCTTCTAGTGCTCCATCCACCCCTCTCTCCACAGACGCACCCGAATTTCTGTCCGTTCCCAAAGATCGGCCCCGGAAAAAGTCTGCCCCAGAAACTCTCACACTTCCAGACCCTGAGAAAAAAGCCACCCTGAATTTACCCGGCATGCACTCTTCAGATAAGCCATGTCGGCCCAAATCTGAGTAACTTTATATAAATATCTCATGGGGTTTTATATTTTCATTTGTTGTTGTTGTTTTTTTTTAAGAATCTTCTGATAGAGAAAAAGACTGCTTTGTCACTCAAACATGTTCCTTCGACCTTTCAGTGTGCATGTGACTCAGTAACTTCACATAGAATATGATTCCCTAAGTATGCTACACAGCATCATATTAGATGTAAGATGTAAGACTTGCAAAGGACAGAAGGAATCTTCTGTAACCACATAGCTGTATGCCAGAGAGGAAGCCTTGTTATTGGGCATTTGATGAGGTTTGGCATGGACTTCAAGGATAAATGAATGAAAACTTTGCACCACTTTTGTTACAAGGTACGGTAGAAAATAGTGAAGTCAGTTTCCTCTCATCAAATCTAAAATTCTCCAAAATACTCTCAGGCATAACATACTTAGCTGTTAAATTTTGAACTGCTAATTACTAATACTTGAATACCAATAGTTACTGAGATTCCTATTTTGTGGTTAGTCTGACTCAGGATTTGGAGCCTAATTAACTCTAAACTTTTGAAAATTTTAATCATCAAGCTATAGAGGCTCCAAGTGCAATTAATAATAACTCATTTATACCTTCCACAGAATTTAATAAAGATTCTACTTGTTTCTGTCTTTTAA PSAT1 CDSATGGACGCCCCCAGGCAGGTGGTCAACTTTGGGCCTGGTCCCGCCAAGCTGCCGCACTCAGTGTTGTTAGAGATACAAAAGGAATTATTAGACTACAAAGGAGTTGGCATTAGTGTTCTTGAAATGAGTCACAGGTCATCAGATTTTGCCAAGATTATTAACAATACAGAGAATCTTGTGCGGGAATTGCTAGCTGTTCCAGACAACTATAAGGTGATTTTTCTGCAAGGAGGTGGGTGCGGCCAGTTCAGTGCTGTCCCCTTAAACCTCATTGGCTTGAAAGCAGGAAGGTGTGCTGACTATGTGGTGACAGGAGCTTGGTCAGCTAAGGCCGCAGAAGAAGCCAAGAAGTTTGGGACTATAAATATCGTTCACCCTAAACTTGGGAGTTATACAAAAATTCCAGATCCAAGCACCTGGAACCTCAACCCAGATGCCTCCTACGTGTATTATTGCGCAAATGAGACGGTGCATGGTGTGGAGTTTGACTTTATACCCGATGTCAAGGGAGCAGTACTGGTTTGTGACATGTCCTCAAACTTCCTGTCCAAGCCAGTGGATGTTTCCAAGTTTGGTGTGATTTTTGCTGGTGCCCAGAAGAATGTTGGCTCTGCTGGGGTCACCGTGGTGATTGTCCGTGATGACCTGCTGGGGTTTGCCCTCCGAGAGTGCCCCTCGGTCCTGGAATACAAGGTGCAGGCTGGAAACAGCTCCTTGTACAACACGCCTCCATGTTTCAGCATCTACGTCATGGGCTTGGTTCTGGAGTGGATTAAAAACAATGGAGGTGCCGCGGCCATGGAGAAGCTTAGCTCCATCAAATCTCAAACAATTTATGAGATTATTGATAATTCTCAAGGATTCTACGTTTGTCCAGTGGAGCCCCAAAATAGAAGCAAGATGAATATTCCATTCCGCATTGGCAATGCCAAAGGAGATGATGCTTTAGAAAAAAGATTTCTTGATAAAGCTCTTGAACTCAATATGTTGTCCTTGAAAGGGCATAGGTCTGTGGGAGGCATCCGGGCCTCTCTGTATAATGCTGTCACAATTGAAGACGTTCAGAAGCTGGCCGCCTTCATGAAAAAATTTTTGGAGATGCATCAGCTATGA PSAT1 3′UTRACACATCCTAACCAGGATATACTCTGTTCTTGAACAACATACAAAGTTTAAAGTAACTTGGGGATGGCTACAAAAAGTTAACACAGTATTTTTCTCAAATGAACATGTTTATTGCAGATTCTTCTTTTTTGAAAGAACAACAGCAAAACATCCACAACTCTGTAAAGCTGGTGGGACCTAATGTCACCTTAATTCTGACTTGAACTGGAAGCATTTTAAGAAATCTTGTTGCTTTTCTAACAAATTCCCGCGTATTTTGCCTTTGCTGCTACTTTTTCTAGTTAGATTTCAAACTTGCCTGTGGACTTAATAATGCAAGTTGCGATTAATTATTTCTGGAGTCATGGGAACACACAGCACAGAGGGTAGGGGGGCCCTCTAGGTGCTGAATCTACACATCTGTGGGGTCTCCTGGGTTCAGCGGCTGTTGATTCAAGGTCAACATTGACCATTGGAGGAGTGGTTTAAGAGTGCCAGGCGAAGGGCAAACTGTAGATCGATCTTTATGCTGTTATTACAGGAGAAGTGACATACTTTATATATGTTTATATTAGCAAGGTCTGTTTTTAATACCATATACTTTATATTTCTATACATTTATATTTCTAATAATACAGTTATCACTGATATATGTAGACACTTTTAGAATTTATTAAATCCTTGACCTTGTGCATTATAGCATTCCATTAGCAAGAGTTGTACCCCCTCCCCAGTCTTCGCCTTCCTCTTTTTAAGCTGTTTTATGAAAAAGACCTAGAAGTTCTTGATTCATTTTTACCATTCTTTCCATAGGTAGAAGAGAAAGTTGATTGGTTGGTTGTTTTTCAATTATGCCATTAAACTAAACATTTCTGTTAAATTACCCTATCCTTTGTTCTCTACTGTTTTCTTTGTAATGTATGACTACGAGAGTGATACTTTGCTGAAAAGTCTTTCCCCTATTGTTTATCTATTGTCAGTATTTTATGTTGAATATGTAAAGAACATTAAAGTCCTAAAACATCTAA PYGB CDSATGGCGAAGCCGCTGACGGACAGCGAGAAGCGGAAGCAGATCAGCGTGCGCGGCCTGGCGGGGCTAGGCGACGTGGCCGAGGTGCGGAAGAGCTTCAACCGGCACTTGCACTTCACGCTGGTCAAGGACCGCAATGTGGCCACGCCCCGCGACTACTTCTTCGCGCTGGCGCACACGGTGCGCGACCACCTCGTGGGCCGCTGGATCCGCACGCAGCAGCACTACTACGAGCGCGACCCCAAGCGCATTTATTATCTTTCCCTGGAATTCTACATGGGTCGCACGCTGCAGAACACGATGGTGAACCTGGGCCTTCAGAATGCCTGCGATGAAGCCATCTATCAGTTGGGGTTAGACTTGGAGGAACTCGAGGAGATAGAAGAAGATGCTGGCCTTGGGAATGGAGGCCTGGGGAGGCTGGCAGCGTGTTTCCTTGACTCAATGGCTACCTTGGGCCTGGCAGCATACGGCTATGGAATCCGCTATGAATTTGGGATTTTTAACCAGAAGATTGTCAATGGCTGGCAGGTAGAGGAGGCCGATGACTGGCTGCGCTACGGCAACCCCTGGGAGAAAGCGCGGCCTGAGTATATGCTTCCCGTGCACTTCTACGGACGCGTGGAGCACACCCCCGACGGCGTGAAGTGGCTGGACACACAGGTGGTGCTGGCCATGCCCTACGACACCCCAGTGCCCGGCTACAAGAACAACACCGTCAACACCATGCGGCTGTGGTCCGCCAAGGCTCCCAACGACTTCAAGCTGCAGGACTTCAACGTGGGAGACTACATCGAGGCGGTCCTGGACCGGAACTTGGCTGAGAACATCTCCAGGGTCCTGTATCCAAATGATAACTTCTTTGAGGGGAAGGAGCTGCGGCTGAAGCAGGAGTACTTCGTGGTGGCCGCCACGCTCCAGGACATCATCCGCCGCTTCAAGTCGTCCAAGTTCGGCTGCCGGGACCCTGTGAGAACCTGTTTCGAGACGTTCCCAGACAAGGTGGCCATCCAGCTGAACGACACCCACCCCGCCCTCTCCATCCCTGAGCTCATGCGGATCCTGGTGGACGTGGAGAAGGTGGACTGGGACAAGGCCTGGGAAATCACGAAGAAGACCTGTGCATACACCAACCACACTGTGCTGCCTGAGGCCTTGGAGCGCTGGCCCGTGTCCATGTTTGAGAAGCTGCTGCCGCGGCACCTGGAGATAATCTATGCCATCAACCAGCGGCACCTGGACCACGTGGCCGCGCTGTTTCCCGGCGATGTGGACCGCCTGCGCAGGATGTCTGTGATCGAGGAGGGGGACTGCAAGCGGATCAACATGGCCCACCTGTGTGTGATTGGGTCCCATGCTGTCAATGGTGTGGCGAGGATCCACTCGGAGATCGTGAAACAGTCGGTCTTTAAGGATTTTTATGAACTGGAGCCAGAGAAGTTCCAGAATAAGACCAATGGCATCACCCCCCGCCGGTGGCTGCTGCTGTGCAACCCGGGGCTGGCCGATACCATCGTGGAGAAAATTGGGGAGGAGTTCCTGACTGACCTGAGCCAGCTGAAGAAGCTGCTGCCGCTGGTCAGTGACGAGGTGTTCATCAGGGACGTGGCCAAGGTCAAACAGGAGAACAAGCTCAAGTTCTCGGCCTTCCTGGAGAAGGAGTACAAGGTGAAGATCAACCCCTCCTCCATGTTCGATGTGCATGTGAAGAGGATCCACGAGTACAAGCGGCAGCTGCTCAACTGCCTGCACGTCGTCACCCTGTACAATCGAATCAAGAGAGACCCGGCCAAGGCTTTTGTGCCCAGGACTGTTATGATTGGGGGCAAGGCAGCGCCCGGTTACCACATGGCCAAGCTGATCATCAAGTTGGTCACCTCCATCGGCGACGTCGTCAATCATGACCCAGTTGTGGGTGACAGGTTGAAAGTGATCTTCCTGGAGAACTACCGTGTGTCCTTGGCTGAGAAAGTGATCCCGGCCGCTGATCTGTCGCAGCAGATCTCCACTGCAGGCACCGAGGCCTCAGGCACAGGCAACATGAAGTTCATGCTCAACGGGGCCCTCACCATCGGCACCATGGACGGCGCCAACGTGGAGATGGCCGAGGAGGCCGGGGCCGAGAACCTCTTCATCTTCGGCCTGCGGGTGGAGGATGTCGAGGCCTTGGACCGGAAAGGGTACAATGCCAGGGAGTACTACGACCACCTGCCCGAGCTGAAGCAGGCCGTGGACCAGATCAGCAGTGGCTTTTTTTCTCCCAAGGAGCCAGACTGCTTCAAGGACATCGTGAACATGCTGATGCACCATGACAGGTTCAAGGTGTTTGCAGACTATGAAGCCTACATGCAGTGCCAGGCACAGGTGGACCAGCTGTACCGGAACCCCAAGGAGTGGACCAAGAAGGTCATCAGGAACATCGCCTGCTCGGGCAAGTTCTCCAGTGACCGGACCATCACGGAGTATGCACGGGAGATCTGGGGTGTGGAGCCCTCCGACCTGCAGATCCCGCCCCCCAACATCCCCCGGGACTAG PYGB 3′UTRGCACACCCTGCCTTGGCGGGACCAGCGGGCATTTGTTTTCTTGCTGACTTTGCACCTCCTTTTTTCCCCAAACACTTTGCCAGCCACTGGTGGTCCCTGCTTTTCTGAGTACCATGTTTCCAGGAGGGGCCATGGGGGTCAGGGTGGTTTTGAGAGAGCAGGGTAAGGAAGGAATGTGCTAGAAGTGCTCCTAGTTTCTTGTAAAGGAAGCCAGAGTTGACAGTACAAAGGGTCGTGGCCAGCCCTGCAGCTTCAGCACCTGCCCCACCCAGAGTGGGAGTCAGGTGGAGCCACCTGCTGGGCTCCCCCAGAACTTTGCACACATCTTGCTATGTATTAGCCGATGTCTTTAGTGTTGAGCCTCTGGATTCTGGGGTCTGGGCCAGTGGCCATAGTGAAGCCTGGGAATGAGTGTTACTGCAGCATCTGGGCTGCCAGCCACAGGGAAGGGCCAAGCCCCATGTAGCCCCAGTCATCCTGCCCAGCCCTGCCTCCTGGCCATGCCGGGAGGGGTCGGATCCTCTAGGCATCGCCTTCACAGCCCCCTGCCCCCTGCCCTCTGTCCTGGCTCTGCACCTGGTATATGGGTCATGGACCCAGATGGGGCTTTCCCTTTGTAGCCATCCAATGGGCATTGTGTGGGTGCTTGGAACCCGGGATGACTGAGGGGGACACTGGAGTGGGTGCTTGTGTCTGCTGTCTCAGAGGCCTTGGTCAGGATGAAGTTGGCTGACACAGCTTAGCTTGGTTTTGCTTATTCAAAAGAGAAAATAACTACACATGGAAATGAAACTAGCTGAAGCCTTTTCTTGTTTTAGCAACTGAAAATTGTACTTGGTCACTTTTGTGCTTGAGGAGGCCCATTTTCTGCCTGGCAGGGGGCAGGTCTGTGCCCTCCCGCTGACTCCTGCTGTGTCCTGAGGTGCATTTCCTGTTGTACACACAAGGGCCAGGCTCCATTCTCCCTCCCTTTCCACCAGTGCCACAGCCTCGTCTGGAAAAAGGACCAGGGGTCCCGGAGGAACCCATTTGTGCTCTGCTTGGACAGCAGGCCTGGCACTGGGAGGTGGGGGTGAGCCCCTCACAGCCTTGCCCCTCCCCAAGGCTGGCAACCTGCCTCCCATTGCCCAAGAGAGAGGGCAGGGAACAGGCTACTGTCCTTCCCTGTGGAATTGCCGAGAAATCTAGCACCTTGCATGCTGGATCTGGGCTGCGGGGAGGCTCTTTTTCTCCCTGGCCTCCAGTGCCCACCAGGAGGATCTGCGCACGGTGCACAGCCCACCAGAGCACTACAGCCTTTTATTGAGTGGGGCAAGTGCTGGGCTGTGGTCGTGCCCTGACAGCATCTTCCCCAGGCAGCGGCTCTGTGGAGGAGGCCATACTCCCCTAGTTGGCCACTGGGGCCACCACCCTGACCACCACTGTGCCCCTCATTGTTACTGCCTTGTGAGATAAAAACTGATTAAACCTTTGTGGCTGTGGTTGGCTGA SHMT2 CDSATGCTGTACTTCTCTTTGTTTTGGGCGGCTCGGCCTCTGCAGAGATGTGGGCAGCTGGTCAGGATGGCCATTCGGGCTCAGCACAGCAACGCAGCCCAGACTCAGACTGGGGAAGCAAACAGGGGCTGGACAGGCCAGGAGAGCCTGTCGGACAGTGATCCTGAGATGTGGGAGTTGCTGCAGAGGGAGAAGGACAGGCAGTGTCGTGGCCTGGAGCTCATTGCCTCAGAGAACTTCTGCAGCCGAGCTGCGCTGGAGGCCCTGGGGTCCTGTCTGAACAACAAGTACTCGGAGGGTTATCCTGGCAAGAGATACTATGGGGGAGCAGAGGTGGTGGATGAAATTGAGCTGCTGTGCCAGCGCCGGGCCTTGGAAGCCTTTGACCTGGATCCTGCACAGTGGGGAGTCAATGTCCAGCCCTACTCCGGGTCCCCAGCCAACCTGGCCGTCTACACAGCCCTTCTGCAACCTCACGACCGGATCATGGGGCTGGACCTGCCCGATGGGGGCCATCTCACCCACGGCTACATGTCTGACGTCAAGCGGATATCAGCCACGTCCATCTTCTTCGAGTCTATGCCCTATAAGCTCAACCCCAAAACTGGCCTCATTGACTACAACCAGCTGGCACTGACTGCTCGACTTTTCCGGCCACGGCTCATCATAGCTGGCACCAGCGCCTATGCTCGCCTCATTGACTACGCCCGCATGAGAGAGGTGTGTGATGAAGTCAAAGCACACCTGCTGGCAGACATGGCCCACATCAGTGGCCTGGTGGCTGCCAAGGTGATTCCCTCGCCTTTCAAGCACGCGGACATCGTCACCACCACTACTCACAAGACTCTTCGAGGGGCCAGGTCAGGGCTCATCTTCTACCGGAAAGGGGTGAAGGCTGTGGACCCCAAGACTGGCCGGGAGATCCCTTACACATTTGAGGACCGAATCAACTTTGCCGTGTTCCCATCCCTGCAGGGGGGCCCCCACAATCATGCCATTGCTGCAGTAGCTGTGGCCCTAAAGCAGGCCTGCACCCCCATGTTCCGGGAGTACTCCCTGCAGGTTCTGAAGAATGCTCGGGCCATGGCAGATGCCCTGCTAGAGCGAGGCTACTCACTGGTATCAGGTGGTACTGACAACCACCTGGTGCTGGTGGACCTGCGGCCCAAGGGCCTGGATGGAGCTCGGGCTGAGCGGGTGCTAGAGCTTGTATCCATCACTGCCAACAAGAACACCTGTCCTGGAGACCGAAGTGCCATCACACCGGGCGGCCTGCGGCTTGGGGCCCCAGCCTTAACTTCTCGACAGTTCCGTGAGGATGACTTCCGGAGAGTTGTGGACTTTATAGATGAAGGGGTCAACATTGGCTTAGAGGTGAAGAGCAAGACTGCCAAGCTCCAGGATTTCAAATCCTTCCTGCTTAAGGACTCAGAAACAAGTCAGCGTCTGGCCAACCTCAGGCAACGGGTGGAGCAGTTTGCCAGGGCCTTCCCCATGCCTGGTTTTGATGAGCATTGASHMT2 3′UTRAGGCACCTGGGAAATGAGGCCCACAGACTCAAAGTTACTCTCCTTCCCCCTACCTGGGCCAGTGAAATAGAAAGCCTTTCTATTTTTTGGTGCGGGAGGGAAGACCTCTCACTTAGGGCAAGAGCCAGGTATAGTCTCCCTTCCCAGAATTTGTAACTGAGAAGATCTTTTCTTTTTCCTTTTTTTGGTAACAAGACTTAGAAGGAGGGCCCAGGCACTTTCTGTTTGAACCCCTGTCATGATCACAGTGTCAGAGACGCGTCCTCTTTCTTGGGGAAGTTGAGGAGTGCCCTTCAGAGCCAGTAGCAGGCAGGGGTGGGTAGGCACCCTCCTTCCTGTTTTTATCTAATAAAATGCTAACCTGCCCTGAGTTTCCATTACTGTGGGTGGGGTTCCCCTGGGCCAAACAGTGATTTGTCTCCCTCAATGTGTACACCGCTCCGCTCCCACCACCGCTACCACAAGGACCCCCGGGGCTGCAGCCTCCTCTTTCTGTCTCTGATCAGAGCCGACACCAGACGTGATTAGCAGGCGCAGCAAATTCAATTTGTTAAATGAAATTGTATTTTG VIPR1 CDSATGCGCCCGCCAAGTCCGCTGCCCGCCCGCTGGCTATGCGTGCTGGCAGGCGCCCTCGCCTGGGCCCTTGGGCCGGCGGGCGGCCAGGCGGCCAGGCTGCAGGAGGAGTGTGACTATGTGCAGATGATCGAGGTGCAGCACAAGCAGTGCCTGGAGGAGGCCCAGCTGGAGAATGAGACAATAGGCTGCAGCAAGATGTGGGACAACCTCACCTGCTGGCCAGCCACCCCTCGGGGCCAGGTAGTTGTCTTGGCCTGTCCCCTCATCTTCAAGCTCTTCTCCTCCATTCAAGGCCGCAATGTAAGCCGCAGCTGCACCGACGAAGGCTGGACGCACCTGGAGCCTGGCCCGTACCCCATTGCCTGTGGTTTGGATGACAAGGCAGCGAGTTTGGATGAGCAGCAGACCATGTTCTACGGTTCTGTGAAGACCGGCTACACCATTGGCTACGGCCTGTCCCTCGCCACCCTTCTGGTCGCCACAGCTATCCTGAGCCTGTTCAGGAAGCTCCACTGCACGCGGAACTACATCCACATGCACCTCTTCATATCCTTCATCCTGAGGGCTGCCGCTGTCTTCATCAAAGACTTGGCCCTCTTCGACAGCGGGGAGTCGGACCAGTGCTCCGAGGGCTCGGTGGGCTGTAAGGCAGCCATGGTCTTTTTCCAATATTGTGTCATGGCTAACTTCTTCTGGCTGCTGGTGGAGGGCCTCTACCTGTACACCCTGCTTGCCGTCTCCTTCTTCTCTGAGCGGAAGTACTTCTGGGGGTACATACTCATCGGCTGGGGGGTACCCAGCACATTCACCATGGTGTGGACCATCGCCAGGATCCATTTTGAGGATTATGGGTGCTGGGACACCATCAACTCCTCACTGTGGTGGATCATAAAGGGCCCCATCCTCACCTCCATCTTGGTAAACTTCATCCTGTTTATTTGCATCATCCGAATCCTGCTTCAGAAACTGCGGCCCCCAGATATCAGGAAGAGTGACAGCAGTCCATACTCAAGGCTAGCCAGGTCCACACTCCTGCTGATCCCCCTGTTTGGAGTACACTACATCATGTTCGCCTTCTTTCCGGACAATTTTAAGCCTGAAGTGAAGATGGTCTTTGAGCTCGTCGTGGGGTCTTTCCAGGGTTTTGTGGTGGCTATCCTCTACTGCTTCCTCAATGGTGAGGTGCAGGCGGAGCTGAGGCGGAAGTGGCGGCGCTGGCACCTGCAGGGCGTCCTGGGCTGGAACCCCAAATACCGGCACCCGTCGGGAGGCAGCAACGGCGCCACGTGCAGCACGCAGGTTTCCATGCTGACCCGCGTCAGCCCAGGTGCCCGCCGCTCCTCCAGCTTCCAAGCCGAAGTCTCCCTGGTCTGA VIPR1 3′UTRCCACCAGGATCCCAGGGGCCCAAGGCGGCCCCTCCCGCCCCTTCCCACTCACCCCGGCAGACGCCGGGGACAGAGGCCTGCCCGGGCGCGGCCAGCCCCGGCCCTGGGCTCGGAGGCTGCCCCCGGCCCCCTGGTCTCTGGTCCGGACACTCCTAGAGAACGCAGCCCTAGAGCCTGCCTGGAGCGTTTCTAGCAAGTGAGAGAGATGGGAGCTCCTCTCCTGGAGGATTGCAGGTGGAACTCAGTCATTAGACTCCTCCTCCAAAGGCCCCCTACGCCAATCAAGGGCAAAAAGTCTACATACTTTCATCCTGACTCTGCCCCCTGCTGGCTCTTCTGCCCAATTGGAGGAAAGCAACCGGTGGATCCTCAAACAACACTGGTGTGACCTGAGGGCAGAAAGGTTCTGCCCGGGAAGGTCACCAGCACCAACACCACGGTAGTGCCTGAAATTTCACCATTGCTGTCAAGTTCCTTTGGGTTAAGCATTACCACTCAGGCATTTGACTGAAGATGCAGCTCACTACCCTATTCTCTCTTTACGCTTAGTTATCAGCTTTTTAAAGTGGGTTATTCTGGAGTTTTTGTTTGGAGAGCACACCTATCTTAGTGGTTCCCCACCGAAGTGGACTGGCCCCTGGGTCAGTCTGGTGGGAGGACGGTGCAACCCAAGGACTGAGGGACTCTGAAGCCTCTGGGAAATGAGAAGGCAGCCACCAGCGAATGCTAGGTCTCGGACTAAGCCTACCTGCTCTCCAAGTCTCAGTGGCTTCATCTGTCAAGTGGGATCTGTCACACCAGCCATACTTATCTCTCTGTGCTGTGGAAGCAACAGGAATCAAGAGCTGCCCTCCTTGTCCACCCACCTATGTGCCAACTGTTGTAACTAGGCTCAGAGATGTGCACCCATGGGCTCTGACAGAAAGCAGATACCTCACCCTGCTACACATACAGGATTTGAACTCAGATCTGTCTGATAGGAATGTGAAAGCACGGACTCTTACTGCTAACTTTTGTGTATCGTAACCAGCCAGATCCTCTTGGTTATTTGTTTACCACTTGTATTATTAATGCCATTATCCCTGAATCCCCCTTGCCACCCCACCCTCCCTGGAGTGTGGCTGAGGAGGCCTCCATCTCATGTATCATCTGGATAGGAGCCTGCTGGTCACAGCCTCCTCTGTCTGCCCTTCACCCCAGTGGCCACTCAGCTTCCTACCCACACCTCTGCCAGAAGATCCCCTCAGGACTGCAACAGGCTTGTGCAACAATAAATGTTGGCTTGGA

MDA-MB-231 cells expressing either a control hairpin or a hairpintargeting miR-126 were transfected with the respective specific reporterconstruct. Thirty hours after transfection, the cells were lysed and theratio of renilla to firefly luciferase expression was determined usingthe dual luciferase assay (E1910, PROMEGA). Cloning primer sequences areshown in Table 2 below.

TABLE 2 Cloning Primers Gene Forward Reverse ABCB9 3′UTRCCGGCCCTCGAGTGGGGGGCCCCTGCTTCTCC CCGGCCGCGGCCGCTTAGGGGTAAGAGGTAGTACABCB9 CDS CCGGCCCTCGAGATGCGGCTGTGGAAGGCGGTCCGGCCGCGGCCGCTCAGGCCTTGTGACTGCCGT IGFBP2 3′UTRCCGGCCCTCGAGACCGCAGCCAGCCGGTGCCT CCGGCCGCGGCCGCTTACTTTTCCTTCCTTTAATIGFBP2 CDS CCGGCCCTCGAGATGCTGCCGAGAGTGGGCTGCCGGCCGCGGCCGCCTACTGCATCCGCTGGGTGT MERTK 3′UTRCCGGCCCTCGAGGGAGAGGTGCGGGGAGACAT CCGGCCGCGGCCGCCTTCCTTATTCATATTTTATMERTK CDS CCGGCCCTCGAGATGGGGCCGGCCCCGCTGCCCCGGCCGCGGCCGCTCACATCAGGACTTCTGAGC PITPNC1 3′UTRCCGGCCCTCGAGCAATGGATGAAGTCCGAGAA CCGGCCGCGGCCGCTTAAAAGACAGAAACAAGTAPITPNC1 CDS CCGGCCCTCGAGATGCTGCTGAAAGAGTACCGCCGGCCGCGGCCGCTCATGTACTTGTTTGGGCAT PSAT1 3′UTRCCGGCCCTCGAGACACATCCTAACCAGGATAT CCGGCCGCGGCCGCTTAGATGTTTTAGGACTTTAPSAT1 CDS CCGGCCGCGGCCGCTCATAGCTGATGCATCTCCACCGGCCCTCGAGATGGACGCCCCCAGGCAGGT PYGB 3′UTRCCGGCCCTCGAGGCACACCCTGCCTTGGCGGG CCGGCCGCGGCCGCTCAGCCAACCACAGCCACAAPYGB CDS CCGGCCGTTTAAACATGGCGAAGCCGCTGACGGACCGGCCGCGGCCGCCTAGTCCCGGGGGATGTTGG SHMT2 3′UTRCCGGCCCTCGAGAGGCACCTGGGAAATGAGGC CCGGCCGCGGCCGCCAAAATACAATTTCATTTAASHMT2 CDS CCGGCCCTCGAGATGCTGTACTTCTCTTTGTTCCGGCCGCGGCCGCTCAATGCTCATCAAAACCAG VIPR CDSCCGGCCGTTTAAACTCAGACCAGGGAGACTTCGG CCGGCCCTCGAGATGCGCCCGCCAAGTCCGCTVIPR1 3′UTR CCGGCCCTCGAGCCACCAGGATCCCAGGGGCCCCGGCCGCGGCCGCTCCAAGCCAACATTTATTGTPotential miR-126 sites in genes were identified by alignment to thecomplementary miR-126 sequence 5-TTACTCACGGTACGA-3, and mutagenesis wasperformed using the QUICKCHANGE Multi Site-Directed Mutagenesis Kit(200514, AGILENT TECHNOLOGIES, Santa Clara, Calif.). Based on the UCSCgenome browser the 3′UTR of MERTK was mutated at position 5 (GTT toCAC), the 3′UTR of IGFBP2 was mutated at position 246 (GGT to CAC), theCDS of PITPNC1 was mutated at position 709 (TAC to GTA) from the startcodon and the CDS of SHMT2 was mutated at position 1126 (GGT to CAC).Mutagenesis primers are in shown in Table 3 below.

TABLE 3 Mutagenesis Primers Gene Forward IGFBP2 3′UTRAAGGGGGTTGTGGTCGGGGAGCTGGCACACAGGTTTGGGGAGGGGGAAGAGAA MERTK 3′UTRATTCTAGGCGATCGCTCGAGGGAGACACGCGGGGAGACATTCCAAAAATCAAG PITPNC1 CDSTATGACAATGGATGATGTTCGGGAAGTAGAGAAAAACATGCATGAACAAACCA SHMT2 CDSGCGAGGCTACTCACTGGTATCAGGTCACACTGACAACCACCTGGTGCTGGTGG ReverseIGFBP2 3′UTR TTCTCTTCCCCCTCCCCAAACCTGTGTGCCAGCTCCCCGACCACAACCCCCTTMERTK 3′UTR CTTGATTTTTGGAATGTCTCCCCGCGTGTCTCCCTCGAGCGATCGCCTAGAATPITPNC1 CDS TGGTTTGTTCATGCATGTTTTTCTCTACTTCCCGAACATCATCCATTGTCATASHMT2 CDS CCACCAGCACCAGGTGGTTGTCAGTGTGACCTGATACCAGTGAGTAGCCTCGC

Cancer Cell Proliferation

LM2 cells (2.5×10⁴) expressing a control hairpin or short hairpinstargeting IGFBP2, PITPNC1 or MERTK were seeded in triplicate in 6 wellplates and viable cells were counted at 5 days after seeding.

Histology

Lungs were prepared by perfusion fixation with 4% paraformaldehydeinfused through the vascular system and through the trachea. Afterexcision, the lungs were placed in 4% paraformaldehyde overnight andembedded in paraffin. Five minutes prior to fixation, 100 mgbiotinylated lectin (B-1175, VECTOR LABORATORIES) was injected into thecirculation via the tail vein. Five-micrometer thick paraffin sectionswere stained with primary antibodies against MECA-32 (DevelopmentalStudies Hybridoma Bank, The University of Iowa, Iowa), Vimentin(VP-V684, VECTOR LABORATORIES) and with FITC labeled Avidin (B-1175,VECTOR LABORATORIES) for the detection of injected biotinylated lectin.Primary antibodies were detected using various Alexa Flourdye-conjugated secondary antibodies. Fluorescence was obtained using aZEISS laser scanning confocal microscope (LSM 510). To determine thevascularisation of metastatic nodules, the MECA-32 and lectin signalswere quantified using IMAGEJ while the metastatic nodules' extents weredetermined through co-staining with human vimentin. The collective areacovered by vessels was determined by subtracting background (rollingball radius of 1 pixel) and by using a pre-determined threshold ascut-off. Vessel density is given as the percentage of area covered bythe blood vessels compared to the total area of the metastatic nodule. Ametastatic nodule was defined by an area positive for vimentin stainingwith a total area above 2000 μm².

Mammary fat pad tumors were excised and submerged into 4%paraformaldehyde for 24 hours. The fixed tissue was embedded in paraffinand sectioned in 5 μm thick slices. Immuno-detection were performedusing antibodies directed towards MECA-32 (Developmental StudiesHybridoma Bank), Mac-2 (CL8942AP, Cederlane, Burlington) and CD45(550539, BD Biosciences). Detection of primary antibodies was performedusing various biotinylated secondary antibodies (Vector Laboratories).The signal was subsequently amplified using the ABC kit (VectorLaboratories), and detected using DAB (3,3′-diaminodbenzidine). Beforemounting the slides were counterstained with hematoxilin.

Dextran permeability was determined as described in Arnold et al., 2010Dis Model Mech 3 (1-2), 57 (2010) with slight modifications. Briefly, anintravenous bolus of 10 mg/ml rhodamine B labeled low molecular weightDextran (1×10⁴ kDa: D1824, INVITROGEN) in sterile PBS was infused.Fifteen minutes later, the mice were anaesthetized and the lungs wereperfused with OCT, removed and frozen on dry ice. Ten-micrometer sectionwas cut and the dextran permeability inside metastatic nodules—asdetermined by vimentin staining—was measured by fluorescence microscopy.Using IMAGEJ, a preset threshold was used to determine the levels ofdextran permeability. The results are presented as the mean percentageof the thresholded area inside the metastatic nodule.

ELISA

IGBFP2 levels in conditioned media were determined using an IGFBP2 ELISA(AAHBLG-1-2, RAYBIOTECH, Norgross, Ga.).

Western Blotting

Cellular lysates from MDA-MB-231 cells were prepared by lysing cells in1 ml ice-cold RIPA buffer containing protease inhibitors (ROCHE,Mannheim, Germany). Conditioned media were prepared by incubatingMDA-MB-231 cells in serum free media for 24 hours. The media was thenconcentrated twenty times by spin filtering. 40 μg protein wassubsequently separated on a 4-12% SDS-PAGE, and transferred to a PVDFmembrane. A monoclonal antibody against human MERTK (CVO-311, CAVEOTHERAPEUTICS, Aurora, Colo.) was used to detect MERTK.

Metastasis Free Survival Analysis

Upon identifying the eight miR-126 regulated genes through anintegrative analysis, it was determined whether the expression of thesegenes in aggregate correlates with human clinical metastasis. Publishedmicroarray data of series from UCSF46, NKI47, and MSKCC13 were used toobtain probe-level expression values. For genes that were represented bymultiple probes, probes that displayed sufficient signal intensity aswell as the highest coefficient of variation (most informative) in anindependent dataset were used. Each breast cancer was classified asmiR-126 signature positive if the sum of the Zscores for the expressionvalues of the 8 genes was greater than the mean of the population.Kaplan-Meier metastasis-free survival curves were generated usingGRAPHPAD PRISM 5 software (GRAPHPAD Software, Inc., LA Jolla, Calif.).Statistical significance for differences between survival curves ofpatients was determined using the Mantel-Cox log-rank test usingGRAPHPAD Prism 5 software.

Vessel Density Analysis

The Kolmogorov-Smirnov test was used to determine the significance ofdifference in the blood vessel density for both MECA-32 and lectinstaining using the publicly available software atphysics.csbsju.edu/stats/KS-test.html.

Example 2 Endogenous Mir-126 Suppressed Systemic Metastatic Colonization

In this example, assays were carried out to analyze metastaticprogression in the setting of miR-126 loss-of-function. This enabled oneto compare in vivo metastatic events between control and miR-126knockdown (KD) cells and to reveal the influence of endogenous miR-126on metastatic colonization.

A MDA-231 breast cancer cell line was generated in which miR-126 wasstably knocked down (94% knock down; FIG. 7) using the miR-Zipanti-sense hairpin microRNA inhibition system. miR-126 KD and control KDcells were injected into immunodeficient mice and evaluated formetastatic colonization capacity in tail-vein colonization assays.miR-126 silencing in poorly metastatic cells increased lung metastaticcolonization by 4.2 fold (P=0.0073) as assessed by quantitativebioluminescence imaging (FIG. 1 a) and dramatically increased metastaticcolonization on gross histology (FIG. 1 a). Intracardiac injection ofMDA miR-126 KD and control KD cells further revealed endogenous miR-126to suppress systemic metastasis as evidenced by enhanced colonization ofmultiple organs such as brain and bone in the setting of miR-126knockdown (FIG. 1 b-c; P=0.0232(b), P=0.0119(c)).

Next, assays were carried out to examine to what extent the dramaticincrease in metastatic colonization observed with miR-126 inhibition wasdue to the effect of miR-126 on tumor growth. To this end, miR-126 KDand control KD cells were injected into the mammary fat pads ofimmunodeficient mice and monitored tumor volume. miR-126 inhibition ledto a modest increase (39.4%) in tumor volume (FIG. 1 d) that was anorder of magnitude smaller than the effect of miR-126 inhibition onmetastasis enhancement—indicating that the effect of miR-126 onmetastasis is not simply a result of its effect on tumor growthsuppression.

To better understand the role of this miRNA on metastatic colonization,the numbers and sizes of all metastases were quantified through imageanalysis of lungs from control and miR-126 KD mice (FIG. 1 e). Thisrevealed a substantial increase in the total number of metastaticnodules in miR-126 KD lungs relative to control lungs (13.6±3.2 versus4.9±1.8; P=0.03). This increase was noted for both small and largenodules (FIG. 1 e) and mirrored the increase in the number of metastasesto other organs (FIG. 1 c). Importantly, the increase in nodule numberwas more pronounced for smaller nodule sizes relative to larger ones,consistent with primarily an increase in the initiation of metastasesrather than an increase in the growth of established metastases. Withoutbeing bound by theory, if miR-126 silencing provides a metastaticinitiation advantage for cells as they initiate metastases in themetastatic niche, its induction in the initial phase of metastasisformation should reduce the number of metastatic nodules. To test this,miR-126 expression was induced in metastatic breast cancer cells (LM2)displaying silencing of this miRNA using a conditional tet-on system.Consistent with this, restoring miR-126 expression to LM2 cells afterthey have extravasated in the lung (Day 3) significantly reducedmetastatic colonization (FIG. 1 f). Thus, restoring miR-126 expressionat this early phase of metastasis initiation in the niche significantlyreduced the number of metastasis nodules visualized at day 49.

The above findings demonstrated that miR-126 silencing enhances theefficiency of metastasis formation leading to a larger number ofmetastases. The findings thus revealed endogenous miR-126 to be asuppressor of metastatic initiation and metastatic colonization.

Example 3 miR-126 Suppresses Metastatic Endothelial Recruitment byBreast Cancer Cells

The above findings suggest that miR-126 silencing can provide metastaticcells and incipient metastases an advantage during metastaticcolonization. While considering the basis of this advantage, it wasnoted that miR-126 knockdown metastases displayed higher vesseldensities on microscopic visualization of lung H&E tissue sections. Toquantify this, co-immunostaining was performed for human vimentin, whichlabels MDA-231 breast cancer cells, and the endothelial marker MECA-32,which allowed one to quantify the endothelial density within metastaticnodules in lungs of mice injected with either control or miR-126 KDbreast cancer cells. Image analysis and quantification revealedmetastases derived from miR-126 KD cells to have a significantly higherendothelial density (FIG. 2 a; 35% increase; P=0.02).

To determine if the enhanced endothelial density in miR-126 KDmetastases represents functional vessels, sugar-binding lectin wasinjected into the circulation of mice prior to lung extractions, andsubsequently stained for the injected lectin. Lectin cytochemistryrevealed that miR-126 knockdown metastases displayed increased densityof functional blood vessels (FIG. 2 b; 33% increase; P=0.001).

Finally, it was sought to determine if miR-126 regulates hemodynamicperfusion to metastases through intravenous perfusion and subsequentvisualization of low-molecular weight dextran (1×10⁴ kDa). Indeed,miR-126 KD metastases displayed significantly increased perfusionrelative to control metastases (FIG. 8; P=0.02).

Thus, these independent and complementary methods reveal that miR-126suppresses in vivo functional metastatic angiogenesis and perfusion.These findings are consistent with miR-126 silencing providingmetastases a selective advantage in angiogenic progression.

Example 4 mir-126 Suppresses Cancer Endothelial Recruitment In Vitro

In this example, it was sought to determine the cellular basis for themiR-126 dependent angiogenesis phenotype observed.

The ability of miR-126 to regulate various cancer-endothelialinteractions such as endothelial adhesion, endothelial proliferation,and tube-formation was analyzed in LM2 metastatic cells (originallyderived from the poorly metastatic MDA-231 population, Minn, A. J. etal., Nature 436 (7050), 518 (2005)) in co-culture with human umbilicalvein endothelial cells (HUVECs). Restoring miR-126 expression to LM2cells, which display silencing of miR-126, did not suppress adhesion ofmetastatic cells to endothelial cells (FIG. 2 c), proliferation ofendothelial cells (FIG. 2 d), or tube formation as assessed by automatedquantification of branch points (FIG. 2 e). Consistent with this,inhibition of miR-126 in MDA-231 cells did not enhance these angiogenicphenotypes either (FIGS. 9 a-c).

The role of miR-126 in regulation of recruitment of endothelial cells tometastatic cells was investigated. Metastatic LM2 cells placed in thebottom of a Boyden chamber strongly recruited HUVECS through a poroustrans-well insert and displayed a significantly enhanced ability torecruit endothelia compared to their poorly metastatic parental line(FIG. 2 f). Endothelial recruitment by metastatic cells was stronglyinhibited (47% reduction) by miR-126 over-expression (FIG. 2 g).Conversely, knockdown of miR-126 in the poorly metastatic parentalMDA-231 population significantly increased endothelial recruitment (146%increase; FIG. 2 g). The CN34LM1a line, a highly lung metastaticderivative that was previously obtained through in-vivo selection of theCN34 primary malignant population (Tavazoie et al., Nature 451 (7175),147 (2008)) (an independent primary malignant population obtained fromthe pleural fluid of a patient with metastatic breast cancer Gupta etal., Nature 446 (7137), 765 (2007)), also displayed significantlyenhanced endothelial recruitment capacity compared to its poorlymetastatic parental line (FIG. 2 h). Both gain- and loss-of-functionexperiments revealed miR-126 to significantly suppress endothelialrecruitment by the CN34 population as well (FIG. 2 i). The findingsreveal enhanced endothelial recruitment capacity to be a key feature ofmetastatic breast cancer populations and identify endogenous miR-126 asa major regulator of this process.

Next, it was sought to determine if endogenous miR-126 can selectivelyregulate endothelial recruitment to breast cancer cells independent oftheir location. Metastatic breast cancer cells expressing a controlhairpin or over-expressing miR-126 were thus implanted into the mammaryfat pads of mice. Metastatic cells, which display silenced miR-126expression, displayed higher vessel density in the mammary glandrelative to poorly metastatic cells. Endothelial recruitment tometastatic cells in the mammary fat pad was inhibited by miR-126expression (FIG. 2 j), while miR-126 knockdown in poorly metastaticcells significantly increased endothelial recruitment to and functionalvessel content of breast tumors growing in mammary fat pads asdetermined by meca-32 staining (FIG. 2 j) and lectin staining (FIG. 10a) respectively. This recruitment effect was selective to endothelialcells as miR-126 silencing did not increase leukocyte density (FIG. 10b) or macrophage density (FIG. 16 c) in mammary tumors.

The above findings revealed that miR-126 selectively regulatesendothelial recruitment to breast cancer cells independent of theiranatomic location.

Example 5 mir-126 Regulon Promotes Endothelial Recruitment

In this example, a systematic search was conducted to identify themolecular targets of miR-126 that mediate endothelial recruitment andmetastatic colonization. Specifically, transcriptomic analysis of LM2cells over-expressing miR-126 was performed and global transcriptalterations to poorly metastatic MDA-231 cells and highly metastatic LM2cells were compared.

Without being bound by theory, it was hypothesized that, given the roleof miR-126 in inhibiting metastasis, the biological mediators of miR-126display increased expression in metastatic cells and that they would besuppressed by this miRNA. A set of 23 genes were identified as they weresuppressed upon miR-126 over-expression (>1.6-fold; FIG. 11; Table 4),and up-regulated (>1.4-fold) in metastatic cells relative to theparental MDA-231 line (FIG. 11).

Of these genes, 14 were validated to be significantly changed byquantitative real-time PCR (qPCR) of MDA-231 control and miR-126 KDcells as well as LM2 control and miR-126 over-expressing cells. Tofurther increase the confidence of this list, the expression of thesegenes in the metastatic derivatives of the independent CN34 line wastested, and 8 genes were identified as displaying significantlyincreased expression in multiple metastatic CN34 derivatives relative totheir parental line (FIG. 3 a).

The contribution of these 8 genes to human metastasis was ascertained bydetermining whether their over-expression in primary human breastcancers correlates with distal metastasis-free survival. Patients whoseprimary breast cancers displayed their over-expression weresignificantly more likely to develop distal metastases and experiencedshorter metastasis-free survival than those whose cancers did notover-express these genes (FIG. 3 b-d). This association displayedsignificance in the UCSF (n=117; P<0.0165), NKI (n=295; P<0.0005), andthe combined MSK/NKI/UCSF cohorts (n=494; P<0.0004). Thus, miR-126suppressed the expression of a set of eight genes that are positivelyand strongly correlated with human metastatic relapse.

Next, assays were carried out to identify the direct targets of miR-126.To this end, the 3′-untranslated regions (3′-UTR's) and coding sequences(CDS's) of all eight miR-126 regulated genes were cloned and used togenerate luciferase fusion constructs. Luciferase reporter assays withthis entire set revealed miR-126 to regulate the expression of IGFBP2and MERTK through interactions with their 3′-UTR's and PITPNC1 and SHMT2through interactions with their coding regions as knockdown ofendogenous miR-126 in MDA-231 cells enhanced expression of theseluciferase fusion genes (FIG. 3 e and FIG. 12). Mutation of miR-126complementary sequences in the 3′-UTR's of IGFBP2 and MERTK abolishedmiR-126 mediated regulation of luciferase expression (FIG. 3 f), whilemutation of the CDS's of PIPNC1 and SHMT2 abolished miRNA mediatedtargeting (FIG. 3 f).

TABLE 4 Fold reduction by miR-126 in LM2 cells Gene Name Fold Gene NameFold Gene Name Fold Gene Name Fold GDF15 −4.15 CTAGE5 −1.93 PRKAR1A−1.80 KIAA0746 −1.71 RARA −3.53 CDA −1.93 CHAC1 −1.80 PADI4 −1.71 P8−2.98 FLJ46385 −1.92 SCD −1.80 BEX2 −1.71 RPS6KA2 −2.54 RALGPS2 −1.92PCK2 −1.80 TAF13 −1.70 C20orf100 −2.47 BDNFOS −1.91 CDC42BPB −1.79 KLF4−1.70 C12orf39 −2.38 MBNL1 −1.91 DSCR1 −1.79 DLG1 −1.70 HERPUD1 −2.37MKX −1.91 TCF7L2 −1.79 DDEFL1 −1.70 CTH −2.36 LPIN1 −1.90 TNRC6C −1.79MID1IP1 −1.70 LOC23117 −2.35 DNAJB9 −1.90 TncRNA −1.78 LOC124220 −1.70LOC23117 −2.35 TncRNA −1.90 CLDN23 −1.78 C10orf58 −1.70 ASNS −2.35BCL2L1 −1.90 GPR153 −1.78 CDKN1C −1.70 RGC32 −2.33 DNAJB9 −1.90 KRTHA4−1.78 DTX3 −1.70 CTH −2.33 ENTH −1.89 SCD −1.78 SETD5 −1.70 NRP1 −2.28S100A5 −1.89 VIPR1 −1.78 SLC7A11 −1.69 RIT1 −2.26 CST4 −1.89 SLC1A4−1.77 WSB1 −1.69 HMGA1 −2.24 TRIB3 −1.89 PNPLA3 −1.77 KIAA1618 −1.69DDIT3 −2.20 PHLDA1 −1.89 PPP1R11 −1.77 PYGB −1.69 MBNL1 −2.20 RGNEF−1.89 CFLAR −1.77 CSNK1A1 −1.69 SUPT6H −2.16 GFPT1 −1.88 NSF −1.77 THBD−1.68 LPIN1 −2.15 TMTC2 −1.88 ABHD4 −1.77 CG012 −1.68 ZNF451 −2.12 TPARL−1.87 SOCS2 −1.77 DDX17 −1.68 THBD −2.10 INHBB −1.87 TACSTD2 −1.76 BGLAP−1.68 ITGB4 −2.10 FASN −1.87 SESN2 −1.76 MAGI1 −1.68 BHLHB8 −2.09 CALB2−1.86 CTNNB1 −1.76 WARS −1.68 SLCO4C1 −2.09 IGFBP2 −1.86 MAP1LC3B −1.76LOC283050 −1.68 AFF4 −2.07 SLC6A9 −1.86 LOC165186 −1.76 AQP3 −1.68ATP6V0D2 −2.05 PLAT −1.86 FLJ20054 −1.75 LOC400581 −1.68 KRT19 −2.05SIN3B −1.86 ZNF69 −1.74 CYLN2 −1.68 SMAD3 −2.04 S100A6 −1.85 TNFSF4−1.74 CD97 −1.68 ARHGAP5 −2.04 WSB1 −1.85 LOC441453 −1.74 CNTNAP3 −1.67DNAJB9 −2.04 C20orf18 −1.85 MARS −1.74 PDE2A −1.67 ATF3 −2.03 HMGCS1−1.85 LOC647135 −1.74 AOF1 −1.67 LOC440092 −2.03 MBNL1 −1.85 ACSL3 −1.74IDS −1.67 RIT1 −2.03 MBNL1 −1.85 SCD −1.74 SCD −1.67 ZNF499 −2.02WHSC1L1 −1.85 SERINC2 −1.73 SHMT2 −1.67 ATXN1 −2.02 NCF2 −1.85 ZCCHC7−1.73 RNF10 −1.67 CST6 −2.01 MERTK −1.84 ETNK1 −1.73 CRLF3 −1.67 WBP2−2.00 PFAAPS −1.84 CHRM3 −1.73 PSAT1 −1.67 ZFAND3 −2.00 RTN4 −1.83DCAMKL1 −1.73 FNBP1 −1.67 FLJ38717 −1.99 LARP6 −1.83 C20orf119 −1.73LOC554203 −1.66 LOC158160 −1.99 TRIB3 −1.83 CDKN1C −1.73 MYADM −1.66PITPNC1 −1.99 RAB37 −1.83 CXorf33 −1.72 ATXN1 −1.66 JMJD1C −1.99LOC399959 −1.83 LPIN1 −1.72 CA12 −1.66 PRO2852 −1.98 SYTL1 −1.82 GEM−1.72 SF3B4 −1.66 AGR2 −1.97 SDF2L1 −1.82 KIAA0746 −1.72 KHDRBS1 −1.66SLC7A5 −1.94 RPH3AL −1.82 LOC115648 −1.72 EGFR −1.66 NSF −1.94 OGDH−1.82 TIA1 −1.72 FRMD5 −1.65 BCL2L1 −1.94 CDYL −1.81 FLJ10120 −1.71ZNF252 −1.65 KIAA1267 −1.93 RHOQ −1.81 DUSP5 −1.71 FNBP1 −1.65 NT5C2−1.93 ITGB4 −1.81 RNF12 −1.71 TNKS2 −1.65 C9orf3 −1.65 C14orf118 −1.61AOF1 −1.65 PIAS1 −1.61 PDP2 −1.65 PXN −1.61 MLLT10 −1.65 C14orf118 −1.61WIRE −1.65 PIAS1 −1.61 ATXN1 −1.65 FLJ43663 −1.65 WARS −1.65 SOS2 −1.61RAB5B −1.64 FLJ43663 −1.60 SQLE −1.64 HCRP1 −1.60 SCNN1A −1.64 LOC646916−1.60 C14orf78 −1.64 NUP43 −1.60 SHMT2 −1.63 PEBP1 −1.60 PSCD3 −1.63FLJ23556 −1.60 LOC643998 −1.63 NRP1 −1.60 PHGDH −1.63 JUP −1.60 HEXA−1.63 CDRT4 −1.63 ACTN4 −1.63 C6orf155 −1.63 EXT1 −1.63 JDP2 −1.63 LSS−1.63 PITPNC1 −1.63 C20orf18 −1.63 CLDN7 −1.63 NPC1 −1.62 IDH1 −1.62THBD −1.62 GSTM4 −1.62 ATP5C1 −1.62 PMM1 −1.62 C9orf5 −1.62 COL8A2 −1.62CST1 −1.62 MAGI1 −1.62 G6PD −1.62 FOSL1 −1.61 RASD1 −1.61 PITX1 −1.61P2RY2 −1.61 HYOU1 −1.61 CSF2RA −1.61 SLC16A4 −1.61 SQLE −1.61 EFHD2−1.61 ABCB9 −1.61 SYDE1 −1.61 MAGI1 −1.61 SLC7A11 −1.61 HSPA5 −1.61

Thus, the binding protein IGF-binding protein 2, the receptor kinaseMERTK, the phosphatidylinositol transfer protein PITPNC1, and thehydroxymethyltranferase enzyme SHMT2 comprise a set of direct targets ofmiR-126 in human breast cancer.

Example 6 IGFBP2, PITPNC1, and MERTK Promote Endothelial Recruitment andMetastasis

In this example, assays were carried out to examine if any of themiR-126 target genes regulate the recruitment of endothelial cells bycancer cells. Of these four genes, knockdown of IGFBP2, MERTK, orPITPNC1 using independent short hairpins significantly suppressed theability of metastatic LM2 cells to recruit endothelial cells (FIG. 4 aand FIG. 13). Importantly, knockdown of these genes did not result in asignificant decrease in cell proliferation (FIG. 14).

Given the robust effects of the miR-126 target genes on endothelialrecruitment, it was examined whether the expression levels of thesegenes individually correlate with metastatic propensity of humancancers. The expression levels of each of these genes were thus analyzedthrough qPCR in an entirely independent set of 96 human breast cancersfor which cDNAs were available.

Patients with stage III and stage IV breast cancers display localmetastatic dissemination and distal metastases, respectively, andcollectively comprise those that develop distal relapse at much higherrates than stage I and II patients. Interestingly, expression levels ofIGFBP2 (P<0.0003), MERTK (P<0.002), and PITPNC1 (P<0.004) wereindividually significantly increased in primary cancers of stage III andIV patients relative to stage I and II patients (FIG. 4 b). Given theirrequirement for endothelial recruitment by metastatic cells, as well astheir direct targeting by miR-126, it was sought to determine if any ofthe miR-126 target genes are required for metastatic colonization.

It was found that, importantly, knockdown of IGFBP2 using independentshort hairpins significantly suppressed metastatic colonization to thelung (sh₁: 10-fold; sh₂: 6.25 fold; FIG. 4 c). In addition, knockdown ofPITPNC1 and MERTK also strongly inhibited metastatic colonization(PITPNC1sh₁: 7.69-fold; PITPNC1sh₂: 4.55-fold, FIG. 4 d; MERTKsh₁:3.91-fold; MERTK1sh₂: 3.08-fold, FIG. 4 e). shRNA sequences used arelisted in Table 5 below.

These findings revealed that the miR-126 direct target genes IGFBP2,PITPNC1 and MERTK are each individually required for endothelialrecruitment and metastatic colonization and individually correlate inexpression with human metastatic progression.

TABLE 5 shRNA sequences Gene Sequence IGFBP2_sh1CCGGCCAGTTCTGACACACGTATTTCTCGAGAAATACGTGTGTCAGAACTGGTTTTT (SEQ ID NO: 1)IGFBP2_sh2 CCGGCAGGTTGCAGACAATGGCGATCTCGAGATCGCCATTGTCTGCAACCTGTTTTT(SEQ ID NO: 2) MERTK_sh1CCGGGCTTCTGGTCTTGATGTATTTCTCGAGAAATACATCAAGACCAGAAGCTTTTT (SEQ ID NO: 3)MERTK_sh2 CCGGCCTGCATACTTACTTACTTTACTCGAGTAAAGTAAGTAAGTATGCAGGTTTTT(SEQ ID NO: 4) PITPNC1_sh1CCGGCGGGTGTATCTCAACAGCAAACTCGAGTTTGCTGTTGAGATACACCCGTTTTTG(SEQ ID NO: 5) PITPNC1_sh2CCGGCAATGGATGAAGTCCGAGAATCTCGAGATTCTCGGACTTCATCCATTGTTTTTG(SEQ ID NO: 6) shSHMT2CCGGCCGGAGAGTTGTGGACTTTATCTCGAGATAAAGTCCACAACTCTCCGGTTTTTG(SEQ ID NO: 7) shcontrolCCGGCAACAAGATGAAGAGCACCAACTC-GAGTTGGTGCTCTTCATCTTGTTGTTTTT(SEQ ID NO: 8)

Example 7 IGFBP2 Mediates Recruitment Through IGF1/IGF1R Activation ofEndothelial Cells

Of the miR-126 targets, IGFBP2 is a secreted factor and, as such, poisedto mediate inter-cellular communication between metastatic cancer cellsand endothelial cells. Thus, it was examined if metastatic cells secreteincreased levels of IGFBP2. It was found that, indeed, ELISA analysisrevealed metastatic LM2 cells to secrete 2.1-fold higher levels of thisfactor than the poorly metastatic MDA-231 parental line (FIG. 5 a).

Members of the IGFBP family exert their effects by interacting withvarious insulin-like growth factors (IGF's) and modulate their bindingto IGF receptors (Baxter, R. C., Horm Res 42 (4-5), 140 (1994) andJones, J. I. et al. Endocr Rev 16 (1), 3 (1995). To determine ifmetastatic endothelial recruitment is mediated through secreted IGFBP2,IGFBP2 binding to the IGF's was inhibited by means of incubation withneutralizing IGFBP2 antibody.

It was found that antibody-mediated inhibition of IGFBP2 in a trans-wellrecruitment assay significantly inhibited metastatic cell endothelialrecruitment to levels comparable to that obtained with miR-126over-expression (FIG. 5 b) and also prevented miR-126 dependentrecruitment (FIG. 5 b). Thus, this effect was specific to themiR-126/IGFBP2 pathway, as inhibition of endothelial recruitment byIGFBP2 antibody was occluded upon miR-126 over-expression (FIG. 5 b).Antibody-mediated inhibition of IGFBP2 also suppressed endothelialrecruitment by the CNLM1A derivative of the independent CN34 malignantline and resulted in a statistically significant reduction inmiR-126-dependent endothelial recruitment (FIG. 5 c). These findingsrevealed secreted IGFBP2 to be an inter-cellular signalling mediator formiR-126-dependent endothelial recruitment by metastatic cells.

IGFBP2 was known to bind both IGF1 and IGF2 in the extracellular spaceand modulate their signaling activity (Jones, J. I. et al. Endocr Rev 16(1), 3 (1995); Arai, T., et al. Endocrinology 137 (11), 4571 (1996);Rajaram, S., et al. Endocr Rev 18 (6), 801 (1997); and Hoflich, A. etal., FEBS Lett 434 (3), 329 (1998)). To determine which IGF mediatesmiR-126-dependent endothelial recruitment, cells were treated withblocking antibodies against IGF1, IGF2, or with immunoglobulin control.Antibody-mediated inhibition of IGF1, but not IGF2, significantlyreduced endothelial recruitment resulting from miR-126 knockdown (FIG. 5d).

Next, it was sought to determine the receptor through which themiR-126-dependent endothelial recruitment is being mediated. Inhibitionof the IGF type-1 receptor (IGF1R) by incubation with IGF1R blockingantibody significantly reduced endothelial recruitment resulting frommiR-126 knockdown, while IGF2R neutralization had no effect (FIG. 5 e).These findings demonstrated that the miR-126/IGFBP2/IGF1 pathwayactivates IGF1R on endothelial cells.

To be certain that the miR-126-dependent recruitment was mediatedthrough IGF1R on endothelial cells—rather than on cancer cells—HUVECendothelial or cancer cells were pre-incubated with the IGF1R antibodyprior to the endothelial recruitment assay. This revealed that onlyIGF1R antibody pre-incubation of endothelial cells inhibited miR-126mediated endothelial recruitment as there was no effect on recruitmentupon pre-incubation with the cancer cells (FIG. 5 f).

The above findings are consistent with metastatic endothelialrecruitment resulting from the secretion of the miR-126 target geneIGFBP2, which binds IGF1 in the extracellular space and enhancesIGF1-dependent activation of the IGF1 receptor on endothelial cells.Enhanced IGF1R activation on endothelial cells in turn stimulatesendothelial migration towards metastatic breast cancer cells. Consistentwith this model, recombinant IGFBP2 protein was sufficient, in adose-dependent way, to promote endothelial chemotaxis (FIG. 5 g) andmigration (FIG. 15) in an IGF1R dependent manner.

Example 8 MERTK Mediates Recruitment Through GAS6

In this example, assays were carried out to investigate the mechanismsby which the other miR-126 target genes PITPNC1 and MERTK mediateendothelial recruitment.

Given the identification of IGFBP2 as a secreted miR-126-dependentfactor that mediates this phenotype, the role PITPNC1 or MERTK in theregulation of the secretion of this factor from cancer cells wasinvestigated. It was found that knockdown of PITPNC1 using independenthairpins reduced IGFBP2 secretion from breast cancer cells (FIG. 6a)—consistent with PITPNC1 regulation of endothelial recruitment beingin part mediated through positive regulation of IGFBP2 secretion.Knockdown of MERTK, however, did not lead to decreased IGFBP2 secretion,suggesting an IGFBP2 independent pathway by which this miR-126 targetgene mediates recruitment.

To determine the mechanism by which the MERTK receptor mediatesrecruitment, assays were carried out to test the impact of its solubleligand GAS6 on cancer-mediated endothelial recruitment. Addingrecombinant GAS6 to the co-culture system—at a physiologicalconcentration found in human serum (Balogh, I. et al., ArteriosclerThromb Vasc Biol 25 (6), 1280 (2005)—potently reduced miR-126 dependentrecruitment (FIG. 6 b), suggesting that GAS6 acts as an inhibitor ofendothelial recruitment. MERTK receptor exists in both membrane boundand soluble forms, where the extracellular domain has been cleaved andthus generally is believed to act as a decoy receptor to negativelyregulate MERTK receptor activation on cells expressing it (Sather, S. etal., Blood (3), 1026 (2007). Soluble MERTK was detected in conditionedmedia of MDA-MB-231 cells (FIG. 16). Without being bound by theory,soluble MERTK released from cancer cells may promote endothelialrecruitment through binding and inhibition of GAS6. Consistent withthis, the addition of recombinant soluble form of the MERTKextracellular domain (MerFc) suppressed exogenous as well as serumGAS6-mediated inhibition of endothelial recruitment by cancer cells(FIG. 6 b). Importantly, this effect was miR-126 dependent (FIG. 6 b).These findings suggest that secreted MERTK from metastatic cells acts asa decoy receptor for GAS6, thereby reducing the suppressive effects ofGAS6 on endothelial cell recruitment. Listed below are amino acidssequences of three GAS6 isoforms.

Isoform 1 MAPSLSPGPAALRRAPQLLLLLLAAECALAALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLDCINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNFFCLCKAGWGGRLCDKDVNECSQENGGCLQICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEARCKNLPGSYSCLCDEGFAYSSQEKACRDVDECLQGRCEQVCVNSPGSYTCHCDGRGGLKLSQDMDTCEDILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA Isoform 2MDTCEDILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA Isoform 3MFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA

To determine whether recombinant forms of IGFBP2 and MERTK, which areexpressed by metastatic cells, and GAS6, which is present in humanserum, are sufficient to regulate endothelial chemotaxis, trans-wellchemotactic assays were performed for quantifying the chemotacticmigration of endothelial cells towards these factors. Recombinant GAS6at low, physiological doses inhibited endothelial chemotaxis towardsrecombinant IGFBP2 (FIG. 6 c). Importantly, recombinant soluble MERTKectodomain abrogated the GAS6 suppressive effect on endothelialchemotaxis (FIG. 6 c). Pre-incubation of endothelial cells with GAS6 didnot affect endothelial migration, suggesting that GAS6 inhibitschemotactic migration. These findings reveal that IGFBP2 mediates apositive migratory and chemotactic signal to endothelial cells throughthe IGF type-1 receptor, while soluble MERTK receptor antagonizes aninhibitory chemotactic signal mediated by GAS6.

Given the roles of IGFPB2, PITPNC1, and MERTK in endothelial recruitmentin vitro and metastatic colonization in vivo, assays were carried out toexamine if these genes regulate in vivo endothelial recruitment. To thisend, MECA-32 staining was performed on lungs from mice injected withcontrol and knockdown breast cancer cells to quantify endothelialrecruitment in vivo as measured by metastatic vessel density. Inhibitingof IGFPB2, PITPNC1, and MERTK individually using independent shorthairpins significantly reduced metastatic endothelial density (FIG. 6 d;P<0.0001 and P=0.002 for shIGFBP2, P=0.01 and P=0.02 for shPITPNC1, andP<0.0001 and P=0.005 for shMERTK). Additionally, lectin perfusion andcytochemistry revealed a significant reduction in functional metastaticvessel content as well (FIG. 17). Thus, the miR-126 target genes IGFBP2,PITPNC1 and MERTK are individually required for metastatic endothelialrecruitment in vivo.

The above findings, comprising both cancer cell mediated endothelialrecruitment and recombinant protein-mediated recruitment assays in vitroas well as in vivo analyses, demonstrated that cancer-expressed IGFBP2and MERTK are necessary and sufficient for mediating endothelialrecruitment and relay parallel pathways emanating from metastatic cancercells (FIG. 6 e).

Example 9 miRNA Regulon that Mediates Metastatic Angiogenesis

The above-described findings revealed that a miRNA expressed in cancercells can non-cell-autonomously regulate the complex process ofmetastatic endothelial recruitment and vascular perfusion through thecoordinate regulation of IGFBP2, MERTK, and PITPNC1—a novel set ofangiogenesis and metastasis genes.

It was found that the increased expression of these metastaticangiogenesis genes endows highly metastatic breast cancer cells withenhanced endothelial recruitment capacity relative to poorly metastaticcells. Metastatic cells over-expressing these genes are able to morereadily establish blood vessels needed for effective colonization.Although the requirement for all of these three genes in metastaticendothelial recruitment was demonstrated, one of them, i.e., secretedIGFBP2, is a trans-cellular mediator of this phenotype.

Additionally, it was discovered the IGF1 signaling pathway—modulated byIGFBP2 secreted from cancer cells and culminating in IGF1R activation onendothelial cells—as a mediator of metastatic-cell endothelialrecruitment and have identified miR-126 in cancer cells as a regulatorof this pathway. Although roles of IGF1 and IGF2 in organismal andcellular growth have been reported (Laviola, L., et al. Curr Pharm Des13 (7), 663 (2007) and Varela-Nieto, I., et al. Curr Pharm Des 13 (7),687 (2007).), the ubiquitous expression of these growth factors andtheir receptors in various tissues and their requirements for normalphysiology limit their therapeutic application (Varela-Nieto, I., et al.Curr Pharm Des 13 (7), 687 (2007)).

IGFBP2 is one of 16 members of the IGFBP family; see Schmid, C., CellBiol Int 19 (5), 445 (1995); Hwa, V., et al. Endocr Rev 20 (6), 761(1999); and Firth, S. M. et al. Endocr Rev 23 (6), 824 (2002).Identification of IGFBP2 as a promoter of metastasis, itsover-expression in metastatic human breast cancer, and the robust effectof its antibody-mediated inhibition on endothelial recruitment bymetastatic cells provides a specific handle for therapeutic targeting ofthe IGF pathway in breast cancer progression and cancer angiogenesis.

While IGFBP2 was identified as a positive regulator of endothelialrecruitment through its activation of a positive regulator of thisprocess (IGF1), MERTK was also discovered as a promoter of recruitmentthrough its inhibition of a negative regulator of endothelial chemotaxis(GAS6). Thus a single miRNA can control a complex phenotype bymodulating both positive and negative regulators of a phenomenon.

Subsequent to its identification as a metastasis suppressor miRNA,miR-126, which is developmentally expressed in endothelial cells, wasgenetically targeted in mice. It was found that miR-126 deletion led topartial embryonic lethality, loss of vascular integrity, and hemorrhage(Wang, S. et al., Dev Cell 15 (2), 261 (2008).). Endothelial-expressedmiR-126 was thus found to be a promoter of normal developmentalangiogenesis in mouse and zebrafish systems (Nicoli, S. et al., Nature464 (7292), 1196 (2010) and Fish, J. E. et al., Dev Cell 15 (2), 272(2008).)

In view of its role as an angiogenesis promoter, it was unexpected thatmiR-126 also suppressed angiogenesis in, e.g., breast cancer, asdisclosed herein. It was unexpected that miR-126 could act in at leasttwo different ways. On one hand, it acts in a cell-type specific fashionto suppress pathologic angiogenesis as disclosed in this application. Asdisclosed in this application, miR-126 suppressed pathologic endothelialmigration to metastases. On the other hand, while in development miR-126expression maintains vessel integrity. Indeed, endothelial miR-126 wasshown to regulate developmental angiogenesis through targeting ofSpred-1 and PIK3R2, genes that were not significantly regulated bymiR-126 in breast cancer cells (Wang, S. et al., Dev Cell 15 (2), 261(2008) and Fish, J. E. et al., Dev Cell 15 (2), 272 (2008).). See Table6. Conversely, it was found that miR-126 inhibition in endothelial cellsdoes not enhance endothelial recruitment by endothelial cells (FIG. 18)as it does in breast cancer cells. Consistent with this, miR-126inhibition in endothelial cells did not alter the expression of PITPNC1,MERTK, or IGFBP2, while it did increase the expression of establishedendothelial miR-126 targets SPRED1 and PIK3R2 (FIG. 19).

TABLE 6 Gene Name LM2 LM2 miR-126 OE Fold SPRED1 979 851 −1.1030 PIK3R22188 1513 −1.2634

Example 10 Identifying Genes or Non-Coding RNAs that Regulate MetastaticCancer Colonization of any Body Tissue

This example describes two approaches for identifying a gene or anon-coding RNA that regulates metastatic cancer colonization of a bodytissue

1. Lenti-miR Approach

Transduction of lenti-miR library into cells and injection into animalsThe lenti-miR library (SYSTEM BIOSCIENCES, Cat # PMIRHPLVAHT-1) was usedin this approach. This library consists of a pool of lentiviruscontaining precursor microRNAs representative of the entire humangenome. Parental populations of the SW620 and LS174T cell-lines (2×10⁵cells) were transduced with the library at a multiplicity of infection(MOI) of 1 to obtain a heterogeneous pool of parental cells withindividual cells over-expressing different microRNA. Each microRNAprecursor was represented at approximately 50× after transduction. Fourdays after transduction, a half-portion of the transduced cells were setaside and genomic DNA extracted using Qiagen DNeasy kit. This was thereference pool of genomic DNA prior to the selective pressure of livercolonization. The remaining half population was injected into the liversof NOD/SCID mice. 3-5 weeks after injection, genomic DNA was extractedfrom the tumors that formed in the livers. Transductions and injectionswere performed in replicates for both cell-lines.

Identification of microRNAs Modulating Liver Colonization

Lenti-miR derived microRNA precursors were recovered from genomic DNA byPCR amplification in the linear range using library-specific, T7promoter-containing primers (forward primer:5′-GAAATTAATACGACTCACTATAGGGCCTGGAGACGCCATCCAC GCTG-3′; reverse primer:5′: GATGTGCGCTCTGCCCACTGAC-3′) on the reference genomic DNA and tumorgenomic DNA. Four PCR reactions using 400 ng of genomic DNA as templatewere performed and pooled per sample to ensure adequate representationof transduced precursor microRNAs.

The resulting PCR amplicons were a composite of different precursormicroRNAs with T7 promoter sequences and were used as templates for invitro transcription to obtain a biotinylated precursor library. Thebiotinylated library obtained from the reference pool and tumors werelabeled with Cy3 and Cy5 respectively and hybridized to a microarraydesigned to detect the microRNA sequences (Genosensor). A dye-swapexperiment was performed to control for dye-bias.

The ratio of the abundance of each microRNA precursor between thereference pool and after selective pressure during liver colonizationwas calculated after normalization of microarray signal. microRNAs thatbecame over-represented in the tumor population compared to thereference pool were considered as promoters and microRNAs that wereunder-represented, suppressors of liver colonization.

2. Lentiplex Approach

Transduction of Lentiplex Library into Cells and Injection into Animals

The lentiplex whole-genome shRNA library (SIGMA-ALDRICH, Cat # SHPH01)was used in this approach. This library is a pooled library oflentivirus containing approximately 150,000 shRNAs targeting the wholehuman genome, with each gene being targeted by 3-5 independent shRNAs.

Parental populations of the cell-lines SW620, LS174T and WiDR (2×10⁶cells) were transduced with the library at a MOI of 1, resulting in apool of heterogeneous population, with individual cells expressing asingle shRNA. Each shRNA was transduced at approximately 100×representation. 48 hrs after transduction; the transduced cells wereselected with puromycin for 48 hrs to remove untransduced cells. Afterantibiotic selection, the remaining cells were allowed to recover for aweek prior to subsequent experiments. A half-portion of the selectedcells were set aside and genomic DNA extracted. This was the referencepool of genomic DNA prior to the selective pressure of livercolonization. The remaining half population was injected into the liversof NOD/SCID mice. 3-5 weeks after injection, genomic DNA was extractedfrom the tumors that formed in the livers. Transductions and injectionswere performed in replicates for all three cell-lines.

Identification of Novel Genes Modulating Liver Colonization ThroughWhole Genome Pooled shRNA Screen

To recover a complex pool of shRNA library sequences from the genomicDNA, a PCR approach followed by Solexa deep sequencing of PCR ampliconswere used. An initial PCR amplification was performed on 500 ng ofgenomic DNA using primers (forward primer:5′-TGGACTATCATATGCTTACCGTAACT-3′; reverse primer: 5′-AAAGAGGATCTCTGTCCCTGT-3′) specific for the virus vector, followed by primers withsequences required for Solexa deep sequencing (forward primer:5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTATTCTTGGCTTTATATATCTTGTGGAAAGGAC-3′; reverse primer: 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGGATGAATACTGCCATTTGTCTCGAGGTCGA-3′) to obtain ampliconscontaining the shRNA sequences. Ten PCR reactions equivalent to 5 ug ofgenomic DNA were performed for each set of genomic DNA and the productspooled for sequencing to ensure adequate representation of shRNAs.

The pooled amplicons represent a composite of genome-wide shRNAsequences and deep sequencing was performed to determine therepresentation of each shRNA species in reference pool compared to thepool amplified from tumors. The count for each shRNA species wasnormalized against the total number of sequences obtained and their genetargets identified by matching to a database provided by Sigma. Genetargets whose shRNAs which became over-represented in the tumor pool areconsidered suppressors of liver colonization and vice versa. To accountfor non-specific effects of shRNA-silencing, only gene targets hitidentified by three or more independent shRNA “hits” were considered asputative suppressors or promoters.

Example 11 Monoclonal Antibody that Neutralizes IGFBP2 FunctionInhibited Endothelial Recruitment by Metastatic Human Breast CancerCells

This example demonstrates a monoclonal antibody that inhibitsendothelial recruitment by metastatic breast cancer cells by binding toIGFBP2 and inhibiting the interaction (binding) of IGF1 to IGFBP2. Byblocking IGF1 binding to IGFBP2, this monoclonal antibody is capable ofinhibiting endothelial recruitment by metastatic human breast cancercells. The methods used to generate neutralizing antibodies to IGFBP2are those commonly known in the art.

In short, mice were immunized with recombinant IGFBP2 total peptide togenerate a polyclonal antibody response. Next, hybridomas libraries weregenerated by fusion of B cells isolated from the immunimized mice tomyeloma cell lines. Supernatant from these hybridomas were then isolatedin order to screen and identify those hybridoma cells generatingantibodies that bind IGFBP2 with high affinity, using antibody capturecompetitive ELISA assays (FIG. 20). Once identified, hybridomasgenerating antibodies with high affinity to IGFBP2 were screened inorder to identify those that generate antibodies capable of inhibitingIGFBP2 from binding IGF1, using antibody capture competitive ELISAassays (FIG. 21). Hybridoma library wo6663-1 contained antibodies thatbound to IGFBP2 to neutralize IGF1 binding, without binding to otherIGFBP family members IGFBP3 and IGFBP4 (FIGS. 20 and 21). To isolatesingle clones (monoclonal) hybridoma cells, seperation and screening wasperformed on hybridoma library wo6663-1. 2000 single hybridoma clones(monoclonal) were screened from this library to identify those thatgenerated monoclonal antibodies that bound to IGFBP2 with high affinityto neutralize IGF1 binding. The table in FIG. 22 lists antibody-captureELISA competitor assay data of several monoclonal antibodies isolatedfrom the above screen, many that had affinity to IGFBP2 and were capableof inhibiting IGF1 binding to IGFBP2 (FIG. 22), including the monoclonalantibody IGFBP2_(—)14 (M14) (dashed box in FIG. 22). These IGFBP2neutralizing monoclonal antibodies were then screened to identify thosecapable of inhibiting endothelial recruitment by metastatic cells usingtrans-well endothelial migration assays. The monoclonal antibodyIGFBP2_M14 (M14) inhibited endothelial recruitment by human metastaticbreast cancer cells:

To identify monoclonal antibodies that could inhibit endothelialrecruitment, the IGFBP2 neutralizing monoclonal antibodies generated inthe above screen were tested in an in vitro endothelial recruitmentassay using transwells. Highly metastatic LM2 human breast cancer cellswere placed in the bottom of a Boyden chamber, where their ability torecruit HUVECS through a porous trans-well insert could be assayed.Small physiologic concentration of IGFBP2 neutralizing antibodies(including M1, M4, M6, M9, M13, M14, M15, and M16 (from FIG. 22)) wereadded to the transwells individually in physiologic concentrations. Ofall antibodies tested, M14 (dashed box in FIG. 22) was able tosignificantly inhibit the recruitment (migrated cells/field) of HUVECcells (50% reduction in migrated cells) versus the negative controlsantibodies IgG and M5 (FIG. 23). This demonstrates the ability of themonoclonal antibody M14 to inhibit human endothelial cell recruitment byhuman metastatic cancer cells (FIG. 23).

To further characterize M14, the heavy chain and light chain variableregions of the antibody were sequenced. The amino acid sequence of boththe heavy chain and light chain variable regions of M14 are presented inTable 7.

TABLE 7 M14 heavy chainQVQLEQSGGGLVQPGGSLKLSCGASGFTFSDYYMYWIRQTPEKRLEWVAYISNG variable regionGGITYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAVYYCVRRSDGSWFVYWamino acid sequence GQGTLVTVSA (SEQ ID NO: 9) M14 light chainDIVITQSPSSLAVSVGEKVTLSCKSSQSLLYSSNQKNCLAWYQQKPGQSPKLLI variable regionYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYLTFGAGTKamino acid sequence LELKRADAAPTVS (SEQ ID NO: 10)

Example 12 Monoclonal Antibody M14 Inhibited Tumor Progression of HumanBreast Cancer In Vivo

This example demonstrates that the IGFBP2 neutralizing antibody M14 iscapable of inhibiting tumor progression and tumor metastasis in vivo ina mouse model of human breast cancer.

To test whether monoclonal antibody M14 was able to reduce tumor burdenand inhibit tumor progression in vivo, 2000 luciferase expressingMDA-MB-231 human breast cancer cells were mixed in a 1:1 ratio withgrowth factor reduced matrigel and injected bilaterally in the mammaryfat pads of NOD-SCID mice. Immediately after injection, luciferin wasinjected and the cancer cell bioluminescence signal was quantified toestablish a Day 0 baseline signal of tumor burden. The mice were thenseparated randomly into two groups: a control group which were treatedwith PBS alone, and an M14 group treated with M14 monoclonal antibody.Intraperitoneal injections of PBS and M14 antibody (250 micrograms) weregiven immediately on Day 0 to mice in each group respectively, and thensubsequently, injections were given biweekly. Tumor burden in both theM14 treated and PBS treated control mice were followed twice a week bybioluminensence from the luciferase reporter. At day 14, tumorprogression was significantly inhibited by treatment with M14 (7 to 11fold reduction in tumor progression) compared with the PBS treated mice(FIG. 24).

The foregoing example and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. All publications cited herein arehereby incorporated by reference in their entirety. As will be readilyappreciated, numerous variations and combinations of the features setforth above can be utilized without departing from the present inventionas set forth in the claims. Such variations are not regarded as adeparture from the scope of the invention, and all such variations areintended to be included within the scope of the following claims.

1-67. (canceled)
 68. A method for treating a cancer in a subject in needthereof, comprising administering to the subject an agent that binds toIGFBP2 and inhibits IGFBP2 from binding to IGF1.
 69. The method of claim68, wherein the treating results in an inhibition of metastasis of thecancer.
 70. The method of claim 68, wherein the cancer is a metastaticcancer.
 71. The method of claim 68, wherein the cancer is breast cancer.72. The method of claim 68, wherein the agent is an antibody, orantigen-binding portion thereof.
 73. The method of claim 68, wherein theagent is an antibody comprising a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 9 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 10, orantigen-binding portion thereof.
 74. The method of claim 68, wherein theagent is an antibody, or antigen-binding portion thereof, which bindsspecifically to the same epitope on IGFBP2 as an antibody comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 9 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 10, or antigen-binding portion thereof.
 75. Themethod of claim 68, wherein the agent is an antibody, or antigen-bindingportion thereof, that competes for IGFBP2 binding with an antibodycomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 9 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 10, or antigen-binding portionthereof.
 76. The method of claim 72, wherein the antibody, orantigen-binding portion thereof, is monoclonal or humanized.
 77. Anantibody comprising a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 9 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10, or antigen-bindingportion thereof.
 78. An antibody, or antigen-binding portion thereof,which binds specifically to the same epitope on IGFBP2 as the antibody,or antigen-binding portion thereof, of claim
 77. 79. An antibody, orantigen-binding portion thereof, that competes for IGFBP2 binding withthe antibody, or antigen-binding portion thereof, of claim
 77. 80. Theantibody, or antigen-binding portion thereof, of claim 77, wherein theantibody, or antigen-binding portion thereof, is monoclonal orhumanized.
 81. A method for treating metastatic cancer in a subject inneed thereof, comprising administering to the subject an agent thatinhibits expression or activity of MERTK.
 82. A method for inhibitingmetastasis of cancer in a subject in need thereof, comprisingadministering to the subject an agent that inhibits expression oractivity of MERTK.
 83. The method of claim 81, wherein the cancer isbreast cancer.
 84. The method of claim 81, wherein the agent is anantibody.
 85. A method for treating metastatic cancer in a subject inneed thereof, comprising administering to the subject an agent thatinhibits expression or activity of PITPNC1.
 86. A method for inhibitingmetastasis of cancer in a subject in need thereof, comprisingadministering to the subject an agent that inhibits expression oractivity of PITPNC1.
 87. The method of claim 85, wherein the cancer isbreast cancer.
 88. The method of claim 85, wherein the agent is anantibody.
 89. The method of claim 85, wherein the agent is a smallmolecule inhibitor.
 90. A method for treating metastatic cancer in asubject in need thereof, comprising administering to the subject anagent that increases expression or activity of GAS6.
 91. A method forinhibiting metastasis of cancer in a subject in need thereof, comprisingadministering to the subject an agent that increases expression oractivity of GAS6.
 92. The method of claim 90, wherein the cancer isbreast cancer.
 93. The method of claim 90, wherein the agent is amonoclonal antibody.
 94. The method of claim 90, wherein the agent isGAS6.
 95. The method of claim 90, wherein the agent is a polypeptidefragment of GAS6.
 96. A method for generating a population of mammaliancancer cells with increased metastatic tissue colonization potential,comprising performing serial rounds of transplantation, isolation, andrepeat transplantation of a population of labeled or unlabeled cancercells into a living tissue.
 97. A method for identifying a gene or anon-coding RNA that regulates metastatic cancer colonization of a bodytissue, comprising, a) introducing one or more shRNA, RNAi, microRNA, ornon-coding RNA molecules into a population of starting cancer cells togenerate a population of engineered cancer cells; b) transplanting saidpopulation of engineered cancer cells into a tissue of the body; c)isolating transplanted cells to obtain a population of isolated cancercells; and d) assessing the quantity of the shRNA, RNAi, microRNA, ornon-coding RNA molecule or protein encoding gene in the population ofisolated cancer cells, wherein a decrease in the quantity of said shRNA,RNAi, microRNA, or non-coding RNA in the population of isolated cellsrelative to its quantity prior to transplantation indicates that thetarget gene or genes of said shRNA, RNAi, microRNA, or non-coding RNAsrepresents a gene required for metastatic colonization of said tissue.98. A method for identifying a gene or a non-coding RNA that regulatesmetastatic cancer colonization of a body tissue, comprising a)introducing one or more DNA molecules that encode a protein encodinggene into a population of starting cancer cells to generate a populationof engineered cancer cells; b) transplanting said population ofengineered cancer cells into a tissue of the body; c) isolatingtransplanted cells to obtain a population of isolated cancer cells; andd) assessing the quantity of the protein encoding gene in the populationof isolated cancer cells, wherein an increase in the quantity of saidprotein encoding gene in the population of isolated cells relative toits quantity prior to transplantation indicates that the proteinencoding gene is required for metastatic colonization of said tissue.99. The method of claim 97, wherein the assessing step is performedusing microarray analysis, DNA sequencing technology, deep sequencingtechnology, or cloning analysis.